Novel conjugates of cc-1065 analogs and bifunctional linkers

ABSTRACT

This invention relates to novel analogs of the DNA-alkylating agent CC-1065 and to their conjugates. Furthermore this invention concerns intermediates for the preparation of said agents and conjugates. The conjugates are designed to release their (multiple) payload after one or more activation steps and/or at a rate and time span controlled by the conjugate in order to selectively deliver and/or controllably release one or more of said DNA alkylating agents. The agents, conjugates, and intermediates can be used to treat an illness that is characterized by undesired (cell) proliferation. As an example, the agents and the conjugates of this invention may be used to treat a tumor.

FIELD OF THE INVENTION

This invention relates to novel analogs of the DNA-alkylating agentCC-1065 and to their conjugates. Furthermore this invention concernsintermediates for the preparation of said agents and conjugates. Theconjugates are designed to release their (multiple) payload after one ormore activation steps and/or at a rate and time span controlled by theconjugate in order to selectively deliver and/or controllably releaseone or more of said DNA-alkylating agents. The agents, conjugates, andintermediates can be used to treat an illness that is characterized byundesired (cell) proliferation. As an example, the agents and theconjugates of this invention may be used to treat a tumor.

BACKGROUND OF THE INVENTION

The duocarmycins, first isolated from a culture broth of Streptomycesspecies, are members of a family of antitumor antibiotics that alsoincludes CC-1065. These extremely potent agents allegedly derive theirbiological activity from an ability to sequence-selectively alkylate DNAat the N3 of adenine in the minor groove, which initiates a cascade ofevents that terminates in an apoptotic cell death mechanism.¹

Although CC-1065 has shown very potent cytotoxicity, it could not beused in the clinic because of serious delayed hepatotoxicity. Thisobservation led to the development of synthetic analogs of CC-1065 (seefor CC-1065 derivatives for example Aristoff et al., J. Org. Chem. 1992,57, 6234; Boger et al., Bioorg. Med. Chem. Lett. 1996, 6, 2207; Boger etal., Chem. Rev. 1997, 97, 787; Milbank et al., J. Med. Chem. 1999, 42,649; Atwell et al., J. Med. Chem. 1999, 42, 3400; Wang et al., J. Med.Chem. 2000, 43, 1541; Boger et al., Bioorg. Med. Chem. Lett. 2001, 11,2021; Parrish et al., Bioorg. Med. Chem. 2003, 11, 3815; Daniell et al.,Bioorg. Med. Chem. Lett. 2005, 15, 177; Tichenor et al., J. Am. Chem.Soc. 2006, 128, 15683; Purnell et al., Bioorg. Med. Chem. 2006, 16,5677; Bando and Sugiyama, Acc. Chem. Res. 2006, 39, 935; Tichenor etal., Nat. Prod. Rep. 2008, 25, 220; MacMillan et al., J. Am. Chem. Soc.2009, 131, 1187; Tietze et al., Anti-Cancer Agents Med. Chem. 2009, 9,304; Gauss et al., Tetrahedron 2009, 65, 6591; Robertson et al., Bioorg.Med. Chem. Lett. 2010, 20, 2722; Boyle et al., Bioorg. Med. Chem. Lett.2010, 20, 1854; Chavda et al., Bioorg. Med. Chem. 2010, 18, 5016; EP0154445; WO 88/04659; WO 90/02746; WO 97/12862; WO 97/32850; WO97/45411; WO 98/52925; WO 99/19298; WO 01/83482; WO 02/067937; WO02/067930; WO 02/068412; WO 03/022806; WO 2004/101767; WO 2006/043839;and WO 2007/051081), which generally showed to have similarcytotoxicity, but reduced hepatotoxicity. Still, however, thesederivatives lack sufficient selectivity for tumor cells, as theselectivity of these agents—and cytotoxic agents in general—is for acertain part based on the difference in the rate of proliferation oftumor cells and normal cells, and therefore they also affect healthycells that show a relatively high proliferation rate. This typicallyleads to severe side effects. Drug concentrations that would completelyeradicate the tumor cannot be reached because of dose-limiting sideeffects such as gastrointestinal tract and bone marrow toxicity. Inaddition, tumors can develop resistance against anticancer agents afterprolonged treatment. In modern drug development, targeting of cytotoxicdrugs to the tumor site can therefore be considered one of the primarygoals.

One promising approach to obtain increased selectivity for tumor cellsor tumor tissue is to exploit the existence of tumor-associatedantigens, receptors, and other receptive moieties, which can serve as atarget. Such a target may be upregulated or to some degree bespecifically present in tumor tissue or in closely associated tissue,such as neovascular tissue, with respect to other tissues in order toachieve efficient targeting. Many targets have been identified andvalidated and several methods to identify and validate targets have beendeveloped.³ By coupling a ligand, e.g. an antibody or antibody fragment,for such a tumor-associated antigen, receptor, or other receptive moietyto a therapeutic agent, this agent can be selectively targeted to tumortissue.

Another promising approach to obtain selectivity for tumor cells ortumor tissue is to exploit the existence of tumor-associated enzymes. Anenzyme that is mainly localized at the tumor site can convert apharmacologically inactive prodrug, which consists of an enzymesubstrate directly or indirectly linked to the toxic drug, to thecorresponding drug in the vicinity of or inside the tumor. Via thisconcept a high concentration of toxic anticancer agent can beselectively generated at the tumor site. All tumor cells may be killedif the dose is sufficiently high, which may decrease development ofdrug-resistant tumor cells.

Enzymes can also be transported to the vicinity of or inside targetcells or target tissue via for example antibody-directed enzyme prodrugtherapy (ADEPT)⁴, polymer-directed enzyme prodrug therapy (PDEPT) ormacromolecular-directed enzyme prodrug therapy (MDEPT)⁵, virus-directedenzyme prodrug therapy (VDEPT)⁶, or gene-directed enzyme prodrug therapy(GDEPT)⁷. With ADEPT, for example, a non-toxic prodrug is selectivelyconverted into a cytotoxic compound at the surface of target cells by anantibody-enzyme conjugate that has been pretargeted to the surface ofthose cells.

Yet another promising approach to obtain selectivity for tumor cells ortumor tissue is to exploit the enhanced permeability and retention (EPR)effect. Through this EPR effect, macromolecules passively accumulate insolid tumors as a consequence of the disorganized pathology ofangiogenic tumor vasculature with its discontinuous endothelium, leadingto hyperpermeability to large macromolecules, and the lack of effectivetumor lymphatic drainage.⁸ By coupling a therapeutic agent directly orindirectly to a macromolecule, said agent can be selectively targeted totumor tissue.

Besides efficient targeting, other important criteria for the successfulapplication of targeted conjugates of cytotoxic agents in tumor therapyare that the one or more agents are released efficiently from theconjugate at the tumor site and that the conjugate is non-cytotoxic oronly very weakly cytotoxic, whereas the cytotoxic agent itself exhibitshighly potent cytotoxicity. Ideally, this leads to the generation ofcytotoxic molecules only at the tumor site, which results in a greatlyincreased therapeutic index with respect to the untargeted cytotoxicagent. Another important criterion for a successful targeted conjugateis that the conjugate must have suitable pharmacological properties,such as sufficient stability in the circulation, low aggregationtendency, and good water solubility. Appropriate water-solubility andhydrophilicity of the drug and/or the linker may contribute to improvedpharmacological properties.

Several conjugates of CC-1065 and derivatives have been described (seefor conjugates of CC-1065 derivatives for example Suzawa et al., Bioorg.Med. Chem. 2000, 8, 2175; Jeffrey et al., J. Med. Chem. 2005, 48, 1344;Wang et al., Bioorg. Med. Chem. 2006, 14, 7854; Tietze et al., Chem.Eur. J. 2007, 13, 4396; Tietze et al., Chem. Eur. J. 2008, 14, 2811;Tietze et al., ChemMedChem 2008, 3, 1946; Lil et al., Tetrahedron Lett.2009, 50, 2932; Tietze et al., Angew. Chem. Int. Ed. 2010, 49, 7336; WO91/16324; WO 94/04535; WO 95/31971; U.S. Pat. No. 5,475,092; U.S. Pat.No. 5,585,499; U.S. Pat. No. 5,646,298; WO 97/07097; WO 97/44000; U.S.Pat. No. 5,739,350; WO 98/11101; WO 98/25898; U.S. Pat. No. 5,843,937;U.S. Pat. No. 5,846,545; WO 02/059122; WO 02/30894; WO 03/086318; WO2005/103040; WO 2005/112919; WO 2006/002895; WO 2006/110476; WO2007/038658; WO 2007/059404; WO 2008/083312; WO 2008/103693; WO2009/026274; WO 2009/064908; WO 2009/073533; WO 2009/073524; WO2009/073546; WO 2009/134977; and US 2009/0162372). In these conjugates,one or more of the favorable properties discussed above may benon-optimal.

Accordingly, there is still a clear need for conjugates of CC-1065derivatives that show a high therapeutic window, contain CC-1065derivatives that have potent cytotoxicity and favorable pharmacologicalproperties, and release the CC-1065 derivatives efficiently.

SUMMARY OF THE INVENTION

The present invention fulfils the above-mentioned need with a compoundof formula (I) or (II):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinDB is a DNA-binding moiety and is selected from the group consisting of

R¹ is a leaving group;R², R^(2′), R³, R^(3′), R⁴, R^(4′), R¹², and R¹⁹ are independentlyselected from H, OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H,C(O)OH, halogen, R^(a), SR^(a), S(O)R^(a), S(O)₂R^(a), S(O)OR^(a),S(O)₂OR^(a), OS(O)R^(a), OS(O)₂R^(a), OS(O)OR^(a), OS(O)₂OR^(a), OR^(a),NHR^(a), N(R^(a))R^(b), ¹N(R^(a))(R^(b))R^(c), P(O)(OR^(a))(OR^(b)),OP(O)(OR^(a)(OR^(b)), SiR^(a)R^(b)R^(c), C(O)R^(a), C(O)OR^(a),C(O)N(R^(a))R^(b), OC(O)R^(a), OC(O)OR^(a), OC(O)N(R^(a))R^(b),N(R^(a))C(O)R^(b), N(R^(a)C(O)OR^(b), and N(R^(a))C(O)N(R^(b))R^(c),wherein

-   -   R^(a), R^(b), and R^(c) are independently selected from H and        optionally substituted C₁₋₃ alkyl or C₁₋₃ hetero alkyl,        or R³+R^(3′) and/or R⁴+R^(4′) are independently selected from        ═O, ═S, ═NOR¹⁸, ═C(R^(18′)) R^(18′), and ═NR¹⁸, R¹⁸ and R^(18′)        being independently selected from H and optionally substituted        C₁₋₃ alkyl, two or more of R², R^(2′), R³, R^(3′), R⁴, R^(4′),        and R¹² optionally being joined by one or more bonds to form one        or more optionally substituted carbocycles and/or heterocycles;        X² is selected from O, C(R¹⁴)(R^(14′)), and NR^(14′), wherein        R¹⁴ and R^(14′) have the same meaning as defined for R⁷ and are        independently selected, or R^(14′) and R^(7′) are absent        resulting in a double bond between the atoms designated to bear        R^(7′) and R^(14′);        R⁵, R^(5′), R⁶, R^(6′), R⁷, and R^(7′) are independently        selected from H, OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂,        C(O)H, C(O)OH, halogen, R^(e), SR^(e), S(O)R^(e), S(O)₂R^(e),        S(O)OR^(e), S(O)₂OR^(e), OS(O)R^(e), OS(O)₂R^(e), OS(O)OR^(e),        OS(O)₂OR^(e), OR^(e), NHR^(e), N(R^(e))R^(f),        N(R^(e))(R^(f))R^(g), P(O)(OR^(e))(OR^(f)),        OP(O)(OR^(e))(OR^(f)), SiR^(e)R^(f)R^(g), C(O)R^(e), C(O)OR^(e),        C(O)N(R^(e))R^(f), OC(O)R^(e), OC(O)OR^(e), OC(O)N(R^(e))R^(f),        N(R^(e))C(O)R^(f), N(R^(e))C(O)OR^(f),        N(R^(e))C(O)N(R^(f))R^(g), and a water-soluble group, wherein    -   R^(e), R^(f), and R^(g) are independently selected from H and        optionally substituted (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₁₅        alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅ cyclo alkyl, C₁₋₁₅        heterocycloalkyl, C₅₋₁₅ aryl, or C₁₋₁₅ heteroaryl, wherein ee is        selected from 1 to 1000, X¹³ is selected from O, S, and NR^(f1),        and R^(f1) and R^(e1) are independently selected from H and C₁₋₃        alkyl, one or more of the optional substituents in R^(e), R^(f),        and/or R^(g) optionally being a water-soluble group, two or more        of R^(e), R^(f), and R^(g) optionally being joined by one or        more bonds to form one or more optionally substituted        carbocycles and/or heterocycles,        or R⁵+R^(5′) and/or R⁶+R^(6′) and/or R⁷+R^(7′) are independently        selected from ═O, ═S, ═NOR^(e3), ═C(R^(e3))R^(e4), and ═NR^(e3),        R^(e3) and R^(e4) being independently selected from H and        optionally substituted C₁₋₃ alkyl, or R^(5′)+R^(6′) and/or        R^(6′)+R^(7′) and/or R^(7′)+R^(14′) are absent, resulting in a        double bond between the atoms designated to bear R^(5′) and        R^(6′), and/or R^(6′) and R^(7′), and/or R^(7′) and R^(14′),        respectively, two or more of R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′),        R¹⁴, and R^(14′) optionally being joined by one or more bonds to        form one or more optionally substituted carbocycles and/or        heterocycles;        X¹ is selected from O, S, and NR¹³, wherein R¹³ is selected from        H and optionally substituted C₁₋₈ alkyl or C₁₋₈ heteroalkyl and        not joined with any other substituent;        X³ is selected from O, S, C(R¹⁵)R^(15′),        —C(R¹⁵)(R^(15′))—C(R¹⁵)(R¹⁵)—, —N(R¹⁵)—N(R^(15′))—,        —C(R¹⁵)(R^(15′))—N(R¹⁵)—, —N(R¹⁵)—C(R¹⁵)(R^(15′))—,        —C(R¹⁵)(R^(15′))—O—, —O—C(R¹⁵)(R^(15′))—, —C(R¹⁵)(R^(15′))—S—,        —S—C(R¹⁵)(R^(15′))—, —C(R¹⁵)═C(R^(15′))—, ═C(R¹⁵)—C(R^(15′))=,        —N═C(R^(15′))—, ═N—C(R^(15′))=, —C(R¹⁵)═N—, ═C(R¹⁵)—N═, —N═N—,        ═N—N═, CR¹⁵, N, and NR¹⁵, or in DB1 and DB2-X³— represents        —X^(3a) and X^(3b)—, wherein X^(3a) is connected to X³⁴, a        double bond is present between X³⁴ and X⁴, and X^(3b) is        connected to X¹¹, wherein X^(3a) is independently selected from        H and optionally substituted (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₈        alkyl, or C₁₋₈ heteroalkyl and not joined with any other        substituent;        X⁴ is selected from O, S, C(R¹⁶)R^(16′), NR¹⁶, N, and CR¹⁶;        X⁵ is selected from O, S, C(R¹⁷)R^(17′), NOR¹⁷, and NR¹⁷,        wherein R¹⁷ and R^(17′) are independently selected from H and        optionally substituted C₁₋₈ alkyl or C₁₋₈ heteroalkyl and not        joined with any other substituent;        X⁶ is selected from CR¹¹, CR¹¹(R^(11′)), N, NR¹¹, O, and S;        X⁷ is selected from CR⁸, CR⁸(R^(8′)), N, NR⁸, O, and S;        X⁸ is selected from CR⁹, CR⁹(R^(9′)), N, NR⁹, O, and S;        X⁹ is selected from CR¹⁰, CR¹⁰(CR^(10′)), N, NR¹⁰, O, and S;        X¹⁰ is selected from CR²⁰, CR²⁰(R^(20′)), N, NR²⁰, O, and S;        X¹¹ is selected from C, CR²¹, and N, or X¹¹-X^(3b) is selected        from CR²¹, CR²¹(R^(21′))N, NR²¹, O, and S;        X¹² is selected from C, CR²², and N;        X⁶*, X⁷*, X⁸*, X⁹*, X¹⁰*, and X¹¹* have the same meaning as        defined for X⁶, X⁷, X⁸, X⁹, X¹⁰, and        X¹¹, respectively, and are independently selected;        X³⁴ is selected from C, CR²³, and N;        the ring B atom of X¹¹* in DB6 and DB7 is connected to a ring        atom of ring A such that ring A and ring B in DB6 and DB7 are        directly connected via a single bond;        =means that the indicated bond may be a single bond or a        non-cumulated, optionally delocalized, double bond;        R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵,        R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′), R²¹,        R^(21′), R²², and R²³ are each independently selected from H,        OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH,        halogen, R^(h), SR^(h), S(O)R^(h), S(O)₂R^(h), S(O)OR^(h),        S(O)₂OR^(h), OS(O)R^(h), OS(O)₂R^(h), OS(O)OR^(h), OS(O)₂OR^(h),        OR^(h), NHR^(h), N(R^(h))R^(i), ⁺N(R^(h))(R^(i))R^(j),        P(O)(OR^(h))(OR^(i)), OP(O)(OR^(h))(OR^(i)), SiR^(h)R^(i)R^(j),        C(O)R^(h), C(O)OR^(h), C(O)N(R^(h))R^(i), OC(O)R^(h),        OC(O)OR^(h), OC(O)N(R^(h))R^(i), N(R^(h))C(O)R^(i),        N(R^(h))C(O)OR^(i), N(R^(h))C(O)N(R^(i))R^(j), and a        water-soluble group, wherein    -   R^(h), R^(i), and R^(j) are independently selected from H and        optionally substituted (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₁₅        alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₁₅        heterocycloalkyl, C₅₋₁₅ aryl, or C₁₋₁₅ heteroaryl, one or more        of the optional substituents in R^(h), R^(i) and/or R^(j)        optionally being a water-soluble group, two or more of R^(h),        R^(i) and R^(j) optionally being joined by one or more bonds to        form one or more optionally substituted carbocycles and/or        heterocycles,        or R⁸+R⁸ and/or R⁹+R^(9′) and/or R¹⁰+R^(10′) and/or R¹¹+R^(11′)        and/or R¹⁵+R^(15′) and/or R¹⁵″+R^(15′″) and/or R¹⁶+R^(16′)        and/or R²⁰+R^(20′) and/or R²¹+R^(21′) are independently selected        from ═O, ═S, ═NOR^(h1), ═C(R^(h1))R^(h2), and ═NR^(h1), R^(h1)        and R^(h2) being independently selected from H and optionally        substituted C₁₋₃ alkyl, two or more of R⁸, R^(8′), R⁹, R^(9′),        R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″),        R¹⁶, R^(16′), R²⁰, R^(20′), R²¹, R^(21′), R²², and R²³        optionally being joined by one or more bonds to form one or more        optionally substituted carbocycles and/or heterocycles;        R^(8b) and R^(9b) are independently selected and have the same        meaning as R⁸, except that they may not be joined with any other        substituent;        one of R⁴ and R^(4′) and one of R¹⁶ and R^(16′) may optionally        be joined by one or more bonds to form one or more optionally        substituted carbocycles and/or heterocycles;        one of R², R^(2′), R³, and R^(3′) and one of R⁵ and R^(5′) may        optionally be joined by one or more bonds to form one or more        optionally substituted carbocycles and/or heterocycles; and        a and b are independently selected from 0 and 1.

In a further aspect, this invention relates to a compound of formula(I′) or (II′):

which is formed through rearrangement of and concomitant elimination ofH—R¹ from the corresponding compounds of formulae (I) and (II), whichare seco compounds (see FIG. 1). Said cyclopropyl ring-containinganalogs are believed to be active species, allegedly being formed fromcompounds of formulae (I) and (II) in vivo via said rearrangement.

In a more specific embodiment, this invention relates to a compound offormula (I) or (II) as described hereinabove, wherein

-   -   a) the DB moiety does not comprise a DA1, DA2, DA1′, or DA2′        moiety; and    -   b) ring B in DB1 is a heterocycle; and    -   c) if X³ in DB1 represents —X^(3a) and X^(3b)— and ring B is        aromatic, then two vicinal substituents on said ring B are        joined to form an optionally substituted carbocycle or        heterocycle fused to said ring B; and    -   d) if X³ in DB2 represents —X^(3a) and X^(3b)— and ring B is        aromatic, then two vicinal substituents on said ring B are        joined to form an optionally substituted heterocycle fused to        said ring B, an optionally substituted non-aromatic carbocycle        fused to said ring B, or a substituted aromatic carbocycle which        is fused to said ring B and to which at least one substituent is        attached that contains a hydroxy group, a primary amino group,        or a secondary amino group, the primary or secondary amine not        being a ring atom in an aromatic ring system nor being part of        an amide; and    -   e) if ring A in DB2 is a 6-membered aromatic ring, then        substituents on ring B are not joined to form a ring fused to        ring B; and    -   f) two vicinal substituents on ring A in DB8 are joined to form        an optionally substituted carbocycle or heterocycle fused to        said ring A to form a bicyclic moiety to which no further rings        are fused; and    -   g) ring A in DB9 together with any rings fused to said ring A        contains at least two ring heteroatoms.

In a further embodiment, this invention relates to a compound of formula(I) or (II) as described hereinabove, wherein at least one of thesubstituents R¹, R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′), R¹⁴, R^(14′), R⁸,R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵, R^(15′), R^(15″),R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′), R²¹, R^(21′), R²², and R²³contains a X¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴ moiety, wherein ff is selectedfrom 1 to 1000 and each X¹⁴ is independently selected from

that is connected to the attachment site of said substituent either viaa direct bond or via a moiety, being part of said same substituent, thatdoes not comprise a disulfide, a hydrazone, a hydrazide, an ester, anatural amino acid, or a peptide containing at least one natural aminoacid, and wherein if ring B in DB1 is an all-carbon ring, X³ is O orNR¹⁵, X⁴ is CH, X³⁴ is C, there is only one X¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴moiety present in said compound of formula (I) or (II) and said moietyis part of R⁶, R⁷, R⁸, R¹⁰, or R¹⁵, then b=1 and ff is ≧5.

A compound of formula (I) or (II) or a conjugate thereof in which ff islarger than 1000 is encompassed by this invention.

In a further embodiment, this invention relates to a compound of formula(I) or (II) as described hereinabove R¹, R⁵, R^(5′), R⁶, R^(6′), R⁷,R^(7′), R¹⁴, R^(14′), R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹,R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′),R²¹, R^(21′), R²², and R²³ contains a triazole moiety.

It is to be understood that if —X³— represents —X^(3a) and X^(3b)— inmoieties DB1 and DB2 these moieties are actually represented by thefollowing structures:

In another aspect, the present invention relates to a conjugate of acompound of formula (I), (II), (I′), or (II′).

In yet another aspect, this invention relates to a compound of formula(III):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinV² is either absent or a functional moiety;each L² is independently absent or a linking group linking V² to L;each L is independently absent or a linking group linking L² to one ormore V¹ and/or Y;each V¹ is independently absent or a conditionally-cleavable orconditionally-transformable moiety, which can be cleaved or transformedby a chemical, photochemical, physical, biological, or enzymaticprocess;each Y is independently absent or a self-eliminating spacer system whichis comprised of 1 or more self-elimination spacers and is linked to V¹,optionally L, and one or more Z;each p and q are numbers representing a degree of branching and are eachindependently a positive integer;z is a positive integer equal to or smaller than the total number ofattachment sites for Z;each Z is independently a compound of formula (I), (II), (I′), or (II′)as defined hereinabove wherein one or more of X¹, R⁵, R^(5′), R⁶,R^(6′), R⁷, R^(7′), R¹⁴, R^(14′), R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′),R¹¹, R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰,R^(20′), R²¹, R^(21′), R²², and R²³ may optionally in addition besubstituted by or be a substituent of formula (V):

wherein each V^(2′), L^(2′), L′, V″, Y′, Z′, p′, q′, and z′ has the samemeaning as defined for V², L², L, V¹, Y, Z, p, q, and z, respectively,and is independently selected, the one or more substituents of formula(V) being independently connected via Y′ to one or more of X¹, R⁵,R^(5′), R⁶, R^(6′), R⁷, R^(7′), R¹⁴, R^(14′), R⁸, R^(8′), R⁹, R^(9′),R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″), R¹⁶,R^(16′), R²⁰, R^(20′), R²¹, R^(21′), R²², and R²³, and/or to one or moreatoms bearing these R substituents; each Z is independently connected toY through either X¹, an atom in R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′), R¹⁴,R^(14′), R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵,R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′), R²¹, R^(21′),R²², R²³, or an atom bearing any of these R substituents; andat least V² or a V¹ is present.

It is noted that in a compound of formula (III), V² or a V¹ needs to bepresent. However, in the one or more moieties of formula (V) that areoptionally present in Z, each V^(2′) and V′ may be independentlyselected to be absent or present.

In a further aspect, this invention relates to a compound of formula(III), wherein

V² is present and selected to be a targeting moiety and there is atleast one group of formula (V) that contains a V^(1′) moiety and eithercomprises a V^(2′), L^(2′), or L moiety that contains aX¹⁴(CH₂CH₂O)_(gg)CH₂CH₂X¹⁴ moiety, wherein gg is selected from 3 to 1000and each X¹⁴ is independently selected from

or said same group of formula (V) comprises at least2X¹⁴CH₂CH₂OCH₂CH₂X¹⁴ moieties, in which each X¹⁴ is independentlyselected.

It is noted that the separate X¹⁴ moieties in the —CH₂CH₂X¹⁴ moietiesthat may be present in a compound of formula (III) are independentlyselected.

It is further noted that z does not represent a degree ofpolymerization; hence z does not indicate that a number of moieties Z orRM2 are connected to one another.

It is further noted that if Y or Y′ is connected to an atom of Z or RM2bearing a specific R substituent instead of to this R substituentitself, this in fact means that this R substituent is absent if this isnecessary to meet valency rules.

It is further noted that if X¹⁴ in for example —CH₂CH₂X¹⁴ represents

, then —CH₂CH₂X¹⁴ should be read as —CH₂CH₂X¹⁴.

The present invention also relates to a compound of formula (IV):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinRM is a reactive moiety and L, V¹, Y, Z, p, and z are as definedhereinabove, except that L is now linking RM to one or more V¹ and/or Y,and V¹, Y, and Z may contain protecting groups, and the one or moreV^(2′)-L^(2′) moieties optionally present in Z as defined hereinabovemay optionally and independently be RM′ instead, which is a reactivemoiety, and wherein, if there is more than 1 reactive moiety in (IV),some or all reactive moieties are the same or different. Theselinker-agent conjugates of formula (IV) may or may not be consideredintermediates for compounds of formula (III).

In a further aspect, the present invention relates to a compound offormula (IV), wherein RM is a reactive moiety selected from carbamoylhalide, acyl halide, active ester, anhydride, α-haloacetyl,α-haloacetamide, maleimide, isocyanate, isothiocyanate, disulfide,thiol, hydrazine, hydrazide, sulfonyl chloride, aldehyde, methyl ketone,vinyl sulfone, halomethyl, and methyl sulfonate, and wherein at leastone group of formula (V), being part of Z, contains a V^(1′) moiety andeither comprises a V^(2′), L^(2′), or L′ moiety that contains aX¹⁴(CH₂CH₂O)_(gg)CH₂CH₂X¹⁴ moiety, wherein gg is selected from 3 to 1000and each X″ is independently selected from

or said same group of formula (V) comprises at least 2X¹⁴CH₂CH₂OCH₂CH₂X¹⁴ moieties, in which each X″ is independentlyselected. These linker-agent conjugates of formula (IV) may or may notbe considered intermediates for compounds of formula (III).

In yet a further aspect, this invention relates to novel bifunctionallinkers that contain a cleavage site, a self-elimination spacer systemand two reactive moieties, one of which can be reacted with atherapeutic or diagnostic moiety, e.g. a compound of formula (II or(II), and the other of which can be reacted with a functional moiety,such as a targeting moiety. These bifunctional linkers can be used toprepare conjugates of formulae (III) and (IV) of this invention orsimilar compounds with different therapeutic or diagnostic moieties.

More specifically, this invention relates to a compound of formula(VIII):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinL, V¹, Y, RM, p, and z are as defined for a compound of formula (IV),and RM2 is a reactive moiety or a leaving group. RM and each RM2 areindependently selected. These bifunctional linkers of formula (VIII) mayor may not be considered intermediates for compounds of formula (III)and (IV).

This invention relates to enantiomerically pure and/ordiastereomerically pure compounds of formulae (I), (II), (III), (IV),and (VIII) as well as to enantiomeric and/or diastereomeric mixtures ofcompounds of formulae (I), (II), (III), (IV), and (VIII). This inventionrelates to pure compounds of formulae (I), (II), (III), (IV), and (VIII)as well as to mixtures of isomers of compounds of formulae (I), (II),(III), (IV), and (VIII).

Compounds of formulae (I) and (II) represent duocarmycin derivativesthat preferably have heteroatoms or polar groups at selected positionsin the DNA-binding moiety or in substituents on the DNA-binding orDNA-alkylating moiety. Compounds of formula (III), which are conjugatesof compounds of formulae (I) and (II), were unexpectedly found to bemore efficacious in vivo and to have improved properties, such asincreased polarity and optimized drug release, compared to similarcompounds from the prior art.

In one embodiment, the current invention relates to a conjugate of acompound of formula (I) or (II) according to one of the aboveembodiments and to derivatives thereof. Such a conjugate contains one ormore promoieties. Advantageously, such a conjugate has a sufficientstability in the circulation, but is efficiently and selectivelyactivated to release the compound of formula (I) or (II) at the targetsite, leading to a suitable therapeutic window. The length and nature ofthe linker between functional moiety and the compound of formula (I) or(II) proved to be an important contributor. In one aspect of thisinvention, the linker has a reduced linker length with respect to linkerlengths in similar conjugates from the prior art, which leads toimproved efficacy. In another aspect, the linker contains aself-elimination spacer system with improved properties, which leads forexample to an optimized self-elimination rate, optimized drug releaseand/or increased polarity. In yet another aspect, the linker betweenfunctional moiety and the compound of formula (I) or (II) contains oneor more groups designed to improve the pharmacokinetic properties of theconjugate. These groups may be present in L and/or Y and/or in any ofthe other moieties making up a compound of formula (III).

Premature release of the parent agent, i.e., the compound of formula (I)or (II), in the circulation may not be desirable, but a relatively fastdeactivation of the released compound might reduce toxic side effects inthis case. Deactivation may be tuned by choosing the appropriateDNA-alkylating and DNA-binding moiety. Deactivation may occur by severalmechanisms, including enzymatic or hydrolytic cleavage of theDNA-alkylating unit from the DNA-binding unit.

Compounds of formulae (I) and (II) are suited for application in drugdelivery purposes, including drug targeting and controlled releaseapplications, using compounds of formulae (III) and (IV).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Rearrangement of a seco compound to a cyclopropyl-containingcompound.

FIG. 2. Graphical illustration of cyclization rates for a series ofcyclization spacer-duocarmycin compounds at 25° C. and pH 7.4 (upperline) and at 37° C. and pH 5 (lower line).

FIG. 3. Human plasma stability for a series of HSA-conjugatedlinker-agent conjugates.

FIG. 4. Single dose efficacy study with Trastuzumab-based antibody-drugconjugates (ADCs) in female nu/nu mice bearing an N87 xenograft.

FIG. 5. Cleavage of N-acetylcysteine-quenched linker-agent conjugates bycathepsin B.

DESCRIPTION OF THE INVENTION

The following detailed description is provided so that the invention maybe more fully understood.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art.

The term “antibody”, as used herein, refers to a full-lengthimmunoglobulin molecule, an immunologically active portion of afull-length immunoglobulin molecule, or a derivative of a full-lengthimmunoglobulin molecule or an active portion thereof, i.e., a moleculethat contains an antigen-binding site that immunospecifically binds anantigen of a target of interest or part thereof, such targets including,but not limited to, tumor cells. A fragment or derivative of afull-length immunoglobulin molecule therefore immunospecifically bindsthe same antigen as said full-length immunoglobulin molecule. Theimmunoglobulin can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, orIgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or subclass.The immunoglobulin, or a derivative or active portion thereof, can bederived from any species, e.g., human, rodent (e.g., mouse, rat, orhamster), donkey, sheep, rabbit, goat, guinea pig, camelid, horse, cow,or chicken, but preferably, it is of human, murine, or rabbit origin, orit is derived from more than one species. Antibodies useful in theinvention include, but are not limited to, monoclonal, polyclonal,bispecific, multispecific, human, humanized, chimeric, and engineeredantibodies, single chain antibodies, Fv fragments, Fd fragments, Fabfragments, F(ab′) fragments, F(ab′)₂ fragments, dAb fragments, fragmentsproduced by a Fab expression library, anti-idiotypic antibodies,isolated CDRs, and epitope-binding fragments of any of the above thatimmunospecifically bind to an antigen-of-interest.

The term “leaving group” refers to a group that can be substituted byanother group in a substitution reaction. Such leaving groups arewell-known in the art, and examples include, but are not limited to, ahalide (fluoride, chloride, bromide, and iodide), azide, a sulfonate(e.g., an optionally substituted C₁₋₆ alkanesulfonate, such asmethanesulfonate, trifluoromethanesulfonate, andtrifluoroethanesulfonate, or an optionally substituted benzenesulfonate,such as p-toluenesulfonate and nosylate), imidazole, a cyclic imidethione, succinimide-N-oxide, phtalimide-N-oxide, p-nitrophenoxide,o-nitrophenoxide, pentafluorophenoxide, tetrafluorophenoxide,1,3,5-trichlorophenoxide, 1,3,5-trifluorophenoxide, a carboxylate, anaminocarboxylate (carbamate), and an alkoxycarboxylate (carbonate). Forsubstitutions at saturated carbon, halides and sulfonates are preferredleaving groups. For substitutions at a carbonyl carbon a halide,succinimide-N-oxide, p-nitrophenoxide, pentafluorophenoxide,tetrafluorophenoxide, a carboxylate, or an alkoxycarboxylate (carbonate)may for example be used as a leaving group. The term “leaving group”also refers to a group that is eliminated as a consequence of anelimination reaction, e.g., an electronic cascade reaction or aspirocyclization reaction. In this instance, a halide, a sulfonate,azide, an amino carboxylate (carbamate) or an alkoxycarboxylate(carbonate) may for example be used as a leaving group. Therefore, anagent or a derivative thereof released from a conjugate through a(multiple) self-elimination is defined as a leaving group according tothis definition. The term “active ester” refers to a functional group inwhich the alkoxy group of the ester moiety is a good leaving group.Examples of such alkoxy groups include, but are not limited to,succinimide-N-oxide, p-nitrophenoxide, pentafluorophenoxide,tetrafluorophenoxide, 1-hydroxybenzotriazole, and1-hydroxy-7-azabenzotriazole, and groups with comparable leavingcapability. Unsubstituted alkyl-based alkoxy groups such as methoxy,ethoxy, isopropoxy, and t-butoxy do not qualify as good leaving groupsand methyl, ethyl, isopropyl, and t-butyl esters are therefore notconsidered to be active esters.

The term “reactive moiety” herein refers to a functional group that canreact with a second functional group under relatively mild conditionsand without the need of prior functionalization of the reactive moiety.The reaction between the reactive moiety and said second functionalgroup will only require the application of some heat, pressure, acatalyst, acid, and/or base. Examples of reactive moieties include, butare not limited to, carbamoyl halide, acyl halide, active ester,anhydride, α-haloacetyl, α-haloacetamide, maleimide, isocyanate,isothiocyanate, disulfide, thiol, hydrazine, hydrazide, sulfonylchloride, aldehyde, methyl ketone, vinyl sulfone, halomethyl, and methylsulfonate.

The term “promoiety” refers to a moiety that is coupled to a compound offormula (I) or (II) to modify its properties and that is to be (partly)removed in vivo from said compound of formula (I) or (II).

The term “water-soluble group” refers to a functional group that is wellsolvated in aqueous environments and that imparts improved watersolubility to the compound to which it is attached. Examples ofwater-soluble groups include, but are not limited to, polyalcohols,straight chain or cyclic saccharides, primary, secondary, tertiary, orquaternary amines and polyamines, sulfate groups, sulfonate groups,sulfinate groups, carboxylate groups, phosphate groups, phosphonategroups, phosphinate groups, ascorbate groups, glycols, includingpolyethylene glycols, and polyethers. Preferred water-soluble groups areprimary, secondary, tertiary, and quaternary amines, carboxylates,phosphonates, phosphates, sulfonates, sulfates,—(CH₂CH₂O)_(yy)CH₂CH₂X¹⁷R^(yy), —(CH₂CH₂O)_(yy)CH₂CH₂X¹⁷—,—X¹⁷(CH₂CH₂O)_(yy)CH₂CH₂—, glycol, oligoethylene glycol, andpolyethylene glycol, wherein yy is selected from 1 to 1000, X¹⁷ isselected from O, S, and NR^(ZZ), and RZZ and RYY are independentlyselected from H and C₁₋₃ alkyl.

The term “substituted”, when used as an adjective to “alkyl”,“heteroalkyl”, “cycloalkyl”, “heterocycloalkyl”, “aryl”, “heteroaryl”,or the like, indicates that said “alkyl”, “heteroalkyl”, “cycloalkyl”,“heterocycloalkyl”, “aryl”, “heteroaryl”, or similar group contains oneor more substituents (introduced by substitution for hydrogen).Exemplary substituents include, but are not limited to, OH, ═O, ═S,═NR^(k), ═N—OR^(k), SH, NH₂, NO₂, NO, N₃, CF₃, CN, OCN, SCN, NCO, NCS,C(O)NH₂, C(O)H, C(O)OH, halogen, R^(k), SR^(k), S(O)R^(k), S(O)OR^(k),S(O)₂R^(k), S(O)₂OR^(k), OS(O)R^(k), OS(O)OR^(k), OS(O)₂R^(k),OS(O)₂OR^(k), S(O)N(R^(k))R¹, OS(O)N(R^(k))R¹, S(O)₂N(R^(k))R¹,OS(O)₂N(R^(k))R¹, OP(O)(OR^(k))(OR¹), P(O)(OR^(k))(OR¹), OR^(k),NHR^(k), N(R^(k))R¹, N(R^(k))(R¹)R^(m), Si(R^(k))(R¹)(R^(m)), C(O)R^(k),C(O)OR^(k), C(O)N(R^(k))R¹, OC(O)R^(k), OC(O)OR^(k), OC(O)N(R^(k))R¹,N(R^(k))C(O)R¹, N(R^(k))C(O)OR¹, N(R^(k))C(O)N(R¹)R^(m), a water-solublegroup, and the thio derivatives of these substituents, and protonated,charged, and deprotonated forms of any of these substituents, whereinR^(k), R^(l), and R^(m) are independently selected from H and optionallysubstituted —(CH₂CH₂O)_(yy)CH₂CH₂X¹⁷R^(Y), C₁₋₁₅ alkyl, C₁₋₁₅heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₁₅ heterocycloalkyl, C₅₋₁₅ aryl, orC₁₋₁₅ heteroaryl, or a combination thereof, wherein yy is selected from1 to 1000, X¹⁷ is independently selected from O, S, and NRZZ, and R^(zz)and RYY are independently selected from H and C₁₋₃ alkyl, two or more ofR^(k), R^(l), and R^(m) optionally being joined by one or more bonds toform one or more optionally substituted carbocycles and/or heterocycles.When there is more than one substituent, each substituent isindependently selected. Two or more substituents may be connected toeach other by replacement of one or more hydrogen atoms on each of thesubstituents by one or more connecting bonds, which may be single,double, or triple bonds, or, if resonance structures are possible, thebond order of said bonds may be different in two or more of theseresonance structures. Two substituents may thus be joined underformation of one or more rings.

When substituents may be “joined by one or more bonds to form one ormore optionally substituted carbocycles and/or heterocycles”, this meansthat the substituents may be connected to each other through replacementof one or more hydrogen atoms on each of the substituents by one or moreconnecting bonds.

The term “aryl” as used herein refers to a carbocyclic aromaticsubstituent comprising 5 to 24 ring carbon atoms, which may be chargedor uncharged and which may consist of one ring or two or more ringsfused together. Examples of aryl groups include, but are not limited to,phenyl, naphthyl, and anthracenyl.

The term “heteroaryl” as used herein refers to a heterocyclic aromaticsubstituent comprising 1 to 24 ring carbon atoms and at least one ringheteroatom, e.g., oxygen, nitrogen, sulfur, silicon, or phosphorus,wherein nitrogen and sulfur may optionally be oxidized and nitrogen mayoptionally be quaternized, which may consist of one ring or two or morerings fused together. Heteroatoms may be directly connected to eachother. Examples of heteroaryl groups include, but are not limited to,pyridinyl, pyrimidyl, furanyl, pyrrolyl, triazolyl, pyrazolyl,pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, thienyl,indolyl, benzofuranyl, benzimidazolyl, benzothiazolyl, purinyl,indazolyl, benzotriazolyl, benzisoxazolyl, quinoxalinyl, isoquinolyl,and quinolyl. In one embodiment, a heteroaryl group comprises 1 to 4heteroatoms. It should be noted that “C₁ heteroaryl group” denotes thatthere is only one carbon present in the ring system of theheteroaromatic group (carbon atoms in optional substituents are thus notcounted). An example of such a heteroaromatic group is a tetrazolylgroup.

“Aryl” and “heteroaryl” groups also encompass ring systems in which oneor more non-aromatic rings are fused to an aryl or heteroaryl ring orring system. The term “alkyl” as used herein refers to a straight chainor branched, saturated or unsaturated hydrocarbyl substituent. Examplesof alkyl groups include, but are not limited to, methyl, ethyl, propyl,butyl, pentyl, hexyl, octyl, decyl, isopropyl, sec-butyl, isobutyl,tert-butyl, isopentyl, 2-methylbutyl, vinyl, allyl, 1-butenyl,2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, and 1-butynyl.

The term “heteroalkyl” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbyl substituent in which atleast one carbon atom is replaced by a heteroatom, e.g., by oxygen,nitrogen, sulfur, silicon, or phosphorus, wherein nitrogen and sulfurmay optionally be oxidized and nitrogen may optionally be quaternized.Heteroatoms may be directly connected to each other. Examples include,but are not limited to, methoxy, ethoxy, propoxy, isopropoxy,n-butyloxy, tert-butyloxy, methyloxymethyl, ethyloxymethyl,methyloxyethyl, ethyloxyethyl, methylaminomethyl, dimethylaminomethyl,methylaminoethyl, dimethylamino ethyl, methylthiomethyl,ethylthiomethyl, ethylthioethyl, and methylthioethyl. The term“cycloalkyl” as used herein refers to a saturated or unsaturatednon-aromatic cyclic hydrocarbyl substituent, which may consist of onering or two or more rings fused together. Examples include, but are notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclo hexadienyl,decalinyl, and 1,4-cyclohexadienyl.

The term “heterocycloalkyl” as used herein refers to a saturated orunsaturated non-aromatic cyclic hydrocarbyl substituent, which mayconsist of one ring or two or more rings fused together, wherein atleast one carbon in one of the rings is replaced by a heteroatom, e.g.,by oxygen, nitrogen, sulfur, silicon, or phosphorus, wherein nitrogenand sulfur may optionally be oxidized and nitrogen may optionally bequaternized. Heteroatoms may be directly connected to each other.Examples include, but are not limited to, tetrahydrofuranyl,pyrrolidinyl, piperidinyl, 1,4-dioxanyl, decahydroquinolinyl,piperazinyl, oxazolidinyl, and morpholinyl. It should be noted that “C₁heterocycloalkyl group” denotes that there is only one carbon present inthe ring system of the heterocycloalkane (carbon atoms in optionalsubstituents are thus not counted). An example of such a group is adioxiranyl group.

The number of carbon atoms that an “alkyl”, “heteroalkyl”, “cycloalkyl”,“heterocycloalkyl”, “aryl”, “heteroaryl”, and the like, may contain isindicated by a designation preceding said terms, i.e., C₁₋₁₀ alkyl meansthat said alkyl may contain from one to ten carbons (carbon atoms inoptional substituents attached to this alkyl are not counted).

The term “carbocycle” herein refers to a saturated or unsaturatedcycloalkane or arene moiety, wherein the terms “cycloalkane” and “arene”are defined as parent moieties of the “cycloalkyl” and “aryl”substituents, respectively, as defined hereinabove.

The term “heterocycle” herein refers to a saturated or unsaturatedheterocycloalkane or heteroarene moiety, wherein the terms“heterocycloalkane” and “heteroarene” are defined as parent moieties ofthe “heterocycloalkyl” and “heteroaryl” substituents, respectively, asdefined hereinabove. The extension “-ylene” as opposed to “-yl” in forexample “alkylene” as opposed to “alkyl” indicates that said for example“alkylene” is a divalent (or multivalent) moiety connected to one ormore other moieties via at least one or more double bonds or two or moresingle bonds, as opposed to being a monovalent group connected to onemoiety via one single bond in said for example “alkyl”. The term“alkylene” therefore refers to a straight chain or branched, saturatedor unsaturated hydrocarbylene moiety; the term “heteroalkylene” as usedherein refers to a straight chain or branched, saturated or unsaturatedhydrocarbylene moiety in which at least one carbon is replaced by aheteroatom; the term “arylene” as used herein refers to a carbocyclicaromatic moiety, which may consist of one ring or two or more ringsfused together; the term “heteroarylene” as used herein refers to acarbocyclic aromatic moiety, which may consist of one ring or two ormore rings fused together, wherein at least one carbon in one of therings is replaced by a heteroatom; the term “cycloalkylene” as usedherein refers to a saturated or unsaturated non-aromatic cyclichydrocarbylene moiety, which may consist of one ring or two or morerings fused together; the term “heterocycloalkylene” as used hereinrefers to a saturated or unsaturated non-aromatic cyclic hydrocarbylenemoiety, which may consist of one ring or two or more rings fusedtogether, wherein at least one carbon in one of the rings is replaced bya heteroatom. Exemplary divalent moieties include those examples givenfor the monovalent groups hereinabove in which one hydrogen atom isremoved.

The prefix “poly” in “polyalkylene”, “polyheteroalkylene”,“polyarylene”, “polyheteroarylene”, polycycloalkylene”,“polyheterocycloalkylene”, and the like, indicates that two or more ofsuch “-ylene” moieties, e.g., alkylene moieties, are joined together toform a branched or unbranched multivalent moiety containing two or moreattachment sites for adjacent moieties. Similarly, the prefix “oligo” infor example oligoethylene glycol indicates that two or more ethyleneglycol moieties are joined together to form a branched or unbranchedmultivalent moiety. The difference between the prefixes “oligo” and“poly” is that the prefix “oligo” is most frequently used to denote arelatively small number of repeating units, while the prefix “poly”usually refers to a relatively large number of repeating units.

Certain compounds of the invention possess chiral centers and/or doublebonds, and/or may have tautomers or atropisomers; the tautomeric,enantiomeric, diastereomeric, atropisomeric, and geometric mixtures oftwo or more isomers, in any composition, as well as the individualisomers (including tautomers and atropisomers) are encompassed withinthe scope of the present invention. Whenever the term “isomer” is used,it refers to an atropisomeric, tautomeric, enantiomeric, diastereomeric,and/or geometric isomer or to a mixture of two or more of these isomers,unless the context dictates otherwise.

The term “peptidomimetic” refers to a group or moiety that has astructure that is different from the general chemical structure of anamino acid or peptide, but functions in a manner similar to a naturallyoccurring amino acid or peptide. Therefore, a peptidomimetic is an aminoacid mimic or peptide mimic.

The term “unnatural amino acid” is intended to represent the Dstereoisomer of a naturally occurring amino acid.

The term “bond” herein refers to a covalent connection between two atomsand may refer to a single bond, a double bond, or a triple bond, or, ifresonance structures are possible, the bond order of said bond may bedifferent in two or more of these resonance structures. For example, ifthe bond is part of an aromatic ring, the bond may be a single bond inone resonance structure and a double bond in another resonancestructure. If it is stated that a “double bond” or “triple bond” ispresent between two atoms, this double, or triple bond may be localized,but it may also be that this double or triple bond is delocalized, whichmeans that only in one or some resonance structures a double or triplebond is indeed present between the two atoms, whereas the bond order maybe different in one or more other resonance structures. At the sametime, bonds marked as single bond in one resonance structure, may bedouble bonds in another resonance structure.

Compounds of the invention may also contain unnatural proportions ofatomic isotopes at one or more atoms that constitute such compounds. Allisotopic variations of compounds of this invention, whether radioactiveor not, are intended to be encompassed within the scope of thisinvention.

The phrase “pharmaceutically active salt” as used herein refers to apharmaceutically acceptable organic or inorganic salt of a compound ofthe invention. For compounds containing one or more basic groups, e.g.,an amine group, acid addition salts can be formed. For compoundscontaining one or more acidic groups, e.g., a carboxylic acid group,base addition salts can be formed. For compounds containing both acidicand basic groups, zwitterions may in addition be obtained as salts. Whenthe compound of the invention comprises more than one charged atom orgroup, there may be multiple (distinct) counterions.

The phrase “pharmaceutically acceptable solvate” refers to anassociation of one or more solvent molecules with a compound of theinvention. Examples of solvents that form pharmaceutically acceptablesolvates include, but are not limited to, water, isopropyl alcohol,ethanol, methanol, DMSO, ethyl acetate, and acetic acid. When referringto water as a solvate, the term “hydrate” can be used.

The term “conjugate” hereinbelow refers to a compound of formula (III)or to a conjugate of a compound of formula (I) or (II) or a derivativethereof, unless the context dictates otherwise. The term “linker-agentconjugate” hereinbelow refers to a compound of formula (IV), unless thecontext dictates otherwise. The term “agent” hereinbelow refers to acompound of formula (I), (II), (I′), or (II′), unless the contextdictates otherwise.

The term “bifunctional linker” hereinbelow refers to a compound offormula (VIII), unless the context dictates otherwise. The term “linker”generally refers to the moiety linking V² to Z in a compound of formula(III) or the promoiety connected to Z in a compound of formula (IV).

The term “core” or “core structure” of a moiety, for example theDNA-binding or DNA-alkylating moiety, refers to the structure thatremains when all R substituents are removed from the formularepresenting said moiety.

The term “targeting moiety” refers to any moiety that specifically bindsor reactively associates or complexes with a moiety specifically or inrelative excess present at or near the target site, on, in, or near thetarget cell, or in (the proximity of) the target tissue or organ, e.g.,a receptor, a receptor complex, substrate, antigenic determinant, orother receptive moiety, or that can target the conjugate to the targetsite via other mechanisms by virtue of its nature, e.g., through the EPReffect. Examples of a targeting moiety include, but are not limited to,an aptamer, an antibody or antibody fragment or derivative, a polymer, adendrimer, a lectin, a biologic response modifier, an enzyme, a vitamin,a growth factor, a steroid, a sugar residue, an oligosaccharide residue,a carrier protein, and a hormone, or any combination thereof.

The phrase “moiety that improves the pharmacological properties of thecompound” refers to a moiety that changes the pharmacological properties(e.g., pharmacodynamic, pharmacokinetic, physicochemical, andbiopharmaceutic properties) of a compound of this invention in such away that a better therapeutic effect can be obtained. The moiety can forexample increase the water solubility, increase the circulation time,increase the therapeutic index, or reduce immunogenicity. The term“linking group” refers to a structural element of a compound that linksone structural element of said compound to one or more other structuralelements of said same compound.

The phrase “a number representing degree of branching” is used to denotethat the subscript number next to a closing bracket represents how manyunits of the moiety within the brackets are each directly attached tothe moiety immediately to the left of the corresponding opening bracket.For example, A-(B)_(b) with b being a number representing a degree ofbranching means that b units B are all directly attached to A. Thismeans that when b is 2, the formula reduces to B-A-B.

The phrase “a number representing degree of polymerization” is used todenote that the subscript number next to a closing bracket representshow many units of the moiety within the brackets are connected to eachother. For example, A-(B)_(b) with b being a number representing adegree of polymerization means that when b is 2, the formula reduces toA-B-B.

The term “single-release spacer” refers to a self-elimination spacerthat can release one moiety upon self-immolation.

The term “multiple-release spacer” refers to a self-elimination spacerthat can release two or more moieties upon (repetitive) self-immolation.

The term “electronic cascade spacer” refers to a self-eliminationspacer, either branched or unbranched, which may self-eliminate throughone or more 1,2+2n electronic cascade eliminations (n≧1).

The term “ω-amino aminocarbonyl cyclization spacer” refers to aself-elimination spacer that may eliminate through a cyclization processunder formation of a cyclic ureum derivative. The term “spacer system”refers to a single self-eliminating spacer moiety or to two or more ofthe same or different self-eliminating spacer moieties coupled together.A spacer system may be branched or unbranched and contain one or moreattachment sites for Z as well as V¹ and optionally L.

In this document and in its claims, the verbs “to comprise”, “to have”,“to contain” and their conjugations are used in their non-limiting senseto mean that items that are “comprised”, “had”, or “contained” areincluded, but items non-specifically mentioned are not excluded. Inaddition, reference to an element by the indefinite article “a” or “an”does not exclude the possibility that more than one of the element ispresent, unless the context clearly requires that there be one and onlyone of the elements. The indefinite article “a” or “an” thus usuallymeans “at least one”.

This specification includes many embodiments. It is noted thatembodiments that are not specifically mentioned and that result fromcombination of two or more of said mentioned embodiments are alsoencompassed by this invention.

In the generic structures throughout this description and in the claimsletters are used to define structural elements. Some of these letterscan be mistaken to represent an atom, such as C, N, O, P, K, B, F, S, U,V, W, I, and Y. To avoid confusion whenever these letters do notrepresent an atom they are given in bold typeface.

When there are one or more adjectives and/or adjective phrases to a nounthat is a) the first in a list of nouns or b) anywhere in the middle ofa list of nouns and said noun and adjectives together are preceded bythe word “and” or “or”, the adjectives do not only bear on said noun,but on all following nouns separately, unless the context dictatesotherwise. This for example means that the phrase “optionallysubstituted C₁₋₄ alkyl, C₁₋₄ heteroalkyl, C₃₋₇ cycloalkyl, or C₁₋₇heterocycloalkyl” should be read as “optionally substituted C₁₋₄ alkyl,optionally substituted C₁₋₄ heteroalkyl, optionally substituted C₃₋₇cycloalkyl, or optionally substituted C₁₋₇ heterocycloalkyl” and thatthe phrase “C₁₋₄ alkyl, C₁₋₄ heteroalkyl, and optionally substitutedC₃₋₇ cycloalkyl, C₅₋₈ aryl, or C₁₋₇ heterocycloalkyl” should be read as“C₁₋₄ alkyl, C₁₋₄ heteroalkyl, and optionally substituted C₃₋₇cycloalkyl, optionally substituted C₅₋₈ aryl, or optionally substitutedC₁₋₇ heterocycloalkyl”.

Throughout this description and in the claims molecular structures orparts thereof are drawn. As usual in such drawings bonds between atomsare represented by lines, in some cases, to indicate stereochemistry, bywavy, bold, or broken or wedged lines. A bond represented by a wavy lineindicates unspecified stereochemistry at the chiral center to which itis connected; a structure or substructure with one or more of such wavybonds in fact represents a set of (sub)structures in which each chiralcenter can either have the R or S configuration. Usually a line endingin space (a “loose” end), i.e., at one end not having another line orspecific atom connected to it, represents a CH₃ group. This is correctfor the drawings representing the compounds of this invention. For thosestructures representing a structural element of the compounds of thisinvention a line ending in space may indicate the position of attachmentof another structural element of the compound. This has been indicatedwith a wavy line perpendicular to and crossing the “loose” line.Furthermore, the structures or parts thereof have been drawn, under theassumption that the structures are read from left to right, meaning thatfor example in the drawings of compounds of formula (III) V² (ifpresent) is located on the left side and Z is located on the right sideof such structures or parts thereof, unless the context impliesotherwise.

The following abbreviations are used herein and have the indicateddefinitions: Ac: acetyl; Bn: benzyl; Boc: tert-butyloxycarbonyl; CBI:1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one; Cbz: carbobenzyloxy;Cit: citrulline; DCC: N,N′-dicyclohexylcarbodiimide; DCE:1,2-dichloroethane; DCM: dichloromethane; DMAP: 4-dimethylaminopyridine;DMF: N,N-dimethylformamide; DMSO: dimethylsulfoxide; DiPEA:N,N-diisopropylethylamine; EEDQ:2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; ESI: electrosprayionization; Fmoc: fluorenylmethyloxycarbonyl; HOBt:1-hydroxy-benzotriazole; HOSu: hydroxysuccinimide; HSA: human serumalbumin; LC/MS: liquid chromatography-mass spectrometry; MOMCl: methylchloromethyl ether; PABA: p-aminobenzyl alcohol; PNPC1: p-nitrophenylchloroformate; RT: room temperature; SEC: size-exclusion chromatography;TCEP: tris(2-carboxyethyl)phosphine; TFA: trifluoroacetic acid; THF:tetrahydrofuran; Val: valine.

Agents, Linker-Agent Conjugates, Conjugates, and Bifunctional Linkers

This invention relates to novel analogs of the DNA-alkylating agentCC-1065. The agents of the present invention are deemed to be used totreat an illness that is characterized by undesired (cell)proliferation. For example, an agent of this invention can be used totreat a tumor, cancer, an autoimmune disease, or an infectious disease.

The conjugates of the present invention are in one aspect deemed to beapplicable to target agents of formulae (I) and (II) to a specifictarget site where the conjugate can be converted into one or more agentsor be induced to be converted into one or more of said agents. Thisinvention can furthermore find application in (non-specific) controlledrelease of one or more of said agents from a conjugate, with the aim offor example enhancing physicochemical, biopharmaceutic, pharmacodynamic,and/or pharmacokinetic properties.

Compounds of formulae (I) and (II) represent duocarmycin derivativesthat preferably have heteroatoms or polar groups at selected positionsin the DNA-binding moiety or in substituents on the DNA-binding orDNA-alkylating moiety. Compounds of formula (III), which are conjugatesof compounds of formulae (I) and (II), were unexpectedly found to bemore efficacious in vivo and to have improved properties, such asincreased polarity and optimized drug release, compared to similarcompounds from the prior art.

In one embodiment, the current invention relates to a conjugate of acompound of formula (I) or (II) according to one of the aboveembodiments and to derivatives thereof. Such a conjugate contains one ormore promoieties. In a more specific embodiment, such a conjugate has asufficient stability in the circulation, but is efficiently andselectively activated to release the compound of formula (I) or (II) atthe target site, leading to a suitable therapeutic window. The lengthand nature of the linker between functional moiety and the compound offormula (I) or (II) proved to be an important contributor. In one aspectof this invention, the linker has a reduced linker length with respectto linker lengths in similar conjugates from the prior art, which leadsto improved efficacy. In another aspect, the linker contains aself-elimination spacer system with improved properties, which leads forexample to an optimized self-elimination rate, optimized drug releaseand/or increased polarity. In yet another aspect, the linker betweenfunctional moiety and the compound of formula (I) or (II) contains oneor more groups designed to improve the pharmacokinetic properties of theconjugate. These groups may be present in L and/or Y and/or in any ofthe other moieties making up a compound of formula (III).Pharmacokinetic properties affected may for example include watersolubility, multi-drug resistance, plasma stability, proteolyticlability, absorption, distribution, metabolism, excretion, andinternalization. Some of these properties may not only affect in vivobehavior, but also in vitro behavior and behavior during the preparationof a compound of formula (III). For example, increased water solubilityof a compound of formula (IV) may favorably affect conjugation of such acompound to a functional moiety in aqueous medium.

Premature release of the parent agent, i.e., the compound of formula (I)or (II), in the circulation may not be desirable, but a relatively fastdeactivation of the released compound might reduce toxic side effects inthis case. Deactivation may be tuned by choosing the appropriateDNA-alkylating and DNA-binding moiety. Deactivation may occur by severalmechanisms, including enzymatic or hydrolytic cleavage of theDNA-alkylating unit from the DNA-binding unit.

Compounds of formulae (I) and (II) are suited for application in drugdelivery purposes, including drug targeting and controlled releaseapplications, using compounds of formulae (III) and (IV).

In a further aspect, this invention relates to novel bifunctionallinkers that contain a cleavage site, a self-elimination spacer systemand two reactive moieties, one of which can be reacted with atherapeutic or diagnostic moiety and the other of which can be reactedwith a functional moiety, such as a targeting moiety. These bifunctionallinkers contain the novel linker elements of this invention and can beused to prepare conjugates of formulae (III) and (IV) of this inventionor similar compounds with different therapeutic or diagnostic moieties.

Agents

In one aspect, the present invention provides a compound of formula (I)or (II):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinDB is a DNA-binding moiety and is selected from the group consisting of

R¹ is a leaving group;R², R^(2′), R³, R^(3′), R⁴, R^(4′), R′², and R¹⁹ are independentlyselected from H, OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H,C(O)OH, halogen, R^(a), SR^(a), S(O)R^(a), S(O)₂R^(a), S(O)OR^(a),S(O)₂OR^(a), OS(O)R^(a), OS(O)₂R^(a), OS(O)OR^(a), OS(O)₂OR^(a), OR^(a),NHR^(a), N(R^(a))R^(b), ⁺N(R^(a))(R^(b))R^(c), P(O)(OR^(a)(OR^(b)),OP(O)(OR^(a)(OR^(b)), SiR^(a)R^(b)R^(c), C(O)R^(a), C(O)OR^(a),C(O)N(R^(a))R^(b), OC(O)R^(a), OC(O)OR^(a), OC(O)N(R^(a))R^(b),N(R^(a))C(O)R^(b), N(R^(a))C(O)OR^(b), and N(R^(a))C(O)N(R^(b))R^(e),wherein

-   -   R^(a), R^(b), and R^(c) are independently selected from H and        optionally substituted C₁₋₃ alkyl or C₁₋₃ hetero alkyl,        or R³+R^(3′) and/or R⁴+R^(4′) are independently selected from        ═O, ═S, ═NOR¹⁸, ═C(R¹⁸) R^(18′), and ═NR¹⁸, R¹⁸ and R^(18′)        being independently selected from H and optionally substituted        C₁₋₃ alkyl, two or more of R², R^(2′), R³, R^(3′), R⁴, R^(4′),        and R¹² optionally being joined by one or more bonds to form one        or more optionally substituted carbocycles and/or heterocycles;        X² is selected from O, C(R¹⁴)(R^(14′)), and NR^(14′), wherein        R¹⁴ and R^(14′) have the same meaning as defined for R⁷ and are        independently selected, or R^(14′) and R^(7′) are absent        resulting in a double bond between the atoms designated to bear        R^(7′) and R^(14′); R⁵, R^(5′), R⁶, R^(6′), R⁷, and R^(7′) are        independently selected from H, OH, SH, NH₂, N₃, NO₂, NO, CF₃,        CN, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(e), SR^(e), S(O)R^(e),        S(O)₂R^(e), S(O)OR^(e), S(O)₂OR^(e), OS(O)R^(e), OS(O)₂R^(e),        OS(O)OR^(e), OS(O)₂OR^(e), OR^(e), NHR^(e), N(R^(e))R^(f),        ⁺N(R^(e))(R^(f))R^(g), P(O)(OR^(e))(OR^(f)),        OP(O)(OR^(e))(OR^(f)), SiR^(e)R^(f)R^(g), C(O)R^(e), C(O)OR^(e),        C(O)N(R^(e))R^(f), OC(O)R^(e), OC(O)OR^(e), OC(O)N(R^(e))R^(f),        N(R^(e))C(O)R^(f), N(R^(e))C(O)OR^(f),        N(R^(e))C(O)N(R^(f))R^(g), and a water-soluble group, wherein    -   R^(e), R^(f), and R^(g) are independently selected from H and        optionally substituted (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₁₅        alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₁₅        heterocycloalkyl, C₅₋₁₅ aryl, or C₁₋₁₅ heteroaryl, wherein ee is        selected from 1 to 1000, X″ is selected from O, S, and NR^(f1),        and R^(f1) and R^(e1) are independently selected from H and C₁₋₃        alkyl, one or more of the optional substituents in R^(e), R^(f),        and/or R^(g) optionally being a water-soluble group, two or more        of R^(e), R^(f), and R^(g) optionally being joined by one or        more bonds to form one or more optionally substituted        carbocycles and/or heterocycles,        or R⁵+R^(5′) and/or R⁶+R^(6′) and/or R⁷+R^(7′) are independently        selected from ═O, ═S, ═NOR^(e3), ═C(R^(e3))R^(e4), and ═NR^(e3),        R^(e3) and R^(e4) being independently selected from H and        optionally substituted C₁₋₃ alkyl, or R^(5′)+R^(6′) and/or        R^(6′)+R^(7′) and/or R^(7′)+R^(14′) are absent, resulting in a        double bond between the atoms designated to bear R^(5′) and        R^(6′), and/or R^(6′) and R^(7′), and/or R^(7′) and R^(14′),        respectively, two or more of R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′),        R¹⁴, and R^(14′) optionally being joined by one or more bonds to        form one or more optionally substituted carbocycles and/or        heterocycles;        X¹ is selected from O, S, and NR¹³, wherein R¹³ is selected from        H and optionally substituted C₁₋₈ alkyl or C₁₋₈ heteroalkyl and        not joined with any other substituent;        X³ is selected from O, S, C(R¹⁵)R^(15′),        —C(R¹⁵)(R^(15′))—C(R¹⁵″)(R^(15′″))—, —N(R¹⁵)—N(R^(15′))—,        —C(R¹⁵)(R^(15′))—N(R¹⁵″)—, —N(R¹⁵″)—C(R¹⁵)(R^(15′))—,        —C(R¹⁵)(R^(15′))—O—, —O—C(R¹⁵)(R^(15′))—, —C(R¹⁵)(R^(15′))—S—,        —S—C(R¹⁵)(R^(15′))—, —C(R¹⁵)═C(R^(15′))—, ═C(R¹⁵)—C(R^(15′))=,        —N═C(R^(15′))—, ═N—C(R^(15′))=, —C(R¹⁵)═N—, ═C(R¹⁵)—N═, —N═N—,        ═N—N═, CR¹⁵, N, and NR¹⁵, or in DB1 and DB2-X³— represents        —X^(3a) and X^(3b)—,        wherein X^(3a) is connected to X³⁴, a double bond is present        between X³⁴ and X⁴, and X^(3b) is connected to X¹¹, wherein        X^(3a) is independently selected from H and optionally        substituted (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₈ alkyl, or C₁₋₈        heteroalkyl and not joined with any other substituent;        X⁴ is selected from O, S, C(R¹⁶)R^(16′), NR¹⁶, N, and CR¹⁶;        X⁵ is selected from O, S, C(R¹⁷)R^(17′), NOR¹⁷, and NR¹⁷,        wherein R¹⁷ and R^(17′) are independently selected from H and        optionally substituted C₁₋₈ alkyl or C₁₋₈ heteroalkyl and not        joined with any other substituent;        X⁶ is selected from CR¹¹, CR¹¹(R^(11′)), N, NR¹¹, O, and S;        X⁷ is selected from CR⁸, CR⁸(R^(8′)), N, NR⁸, O, and S;        X⁸ is selected from CR⁹, CR⁹(R^(9′)), N, NR⁹, O, and S;        X⁹ is selected from CR¹⁰, CR¹⁰R^(10′)), N, NR¹⁰, O, and S;        X¹⁰ is selected from CR²⁰, CR²⁰(R^(20′)), N, NR²⁰, O, and S;        X¹¹ is selected from C, CR²¹, and N, or X¹¹-X^(3b) is selected        from CR²¹, CR²¹(R^(21′)), N, NR²¹, o, and S;        X¹² is selected from C, CR²², and N;        X⁶*, X⁷*, X⁸*, X⁹*, X¹⁰*, and X¹¹* have the same meaning as        defined for X⁶, X⁷, X⁸, X⁹, X¹⁰, and        X¹¹, respectively, and are independently selected;        X³⁴ is selected from C, CR²³, and N;        the ring B atom of X¹¹* in DB6 and DB7 is connected to a ring        atom of ring A such that ring A and ring B in DB6 and DB7 are        directly connected via a single bond;        means that the indicated bond may be a single bond or a        non-cumulated, optionally delocalized, double bond;        R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵,        R^(15′), R¹⁵″, R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′), R²¹,        R^(21′), R²², and R²³ are each independently selected from H,        OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH,        halogen, R^(h), SR^(h), S(O)R^(h), S(O)₂R^(h), S(O)OR^(h),        S(O)₂OR^(h), OS(O)R^(h), OS(O)₂R^(h), OS(O)OR^(h), OS(O)₂OR^(h),        OR^(h), NHR^(h), N(R^(h))R^(i), ⁺N(R^(h))(R^(i))R^(j),        P(O)(OR^(h))(OR^(i)), OP(O)(OR^(h))(OR^(h)), SiR^(h)R^(i)R^(j),        C(O)R^(h), C(O)OR^(h), C(O)N(R^(h))R^(i), OC(O)R^(h),        OC(O)OR^(h), OC(O)N(R^(h))R^(i), N(R^(h))C(O)R^(i),        N(R^(h))C(O)OR^(i), N(R^(h))C(O)N(R^(i))RJ, and a water-soluble        group, wherein    -   R^(h), and RJ are independently selected from H and optionally        substituted (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₁₅ alkyl, C₁₋₁₅        heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₁₅ heterocycloalkyl, C₅₋₁₅        aryl, or C₁₋₁₅ heteroaryl, one or more of the optional        substituents in R^(h), R^(i), and/or R optionally being a        water-soluble group, two or more of R^(h), R^(i), and R^(j)        optionally being joined by one or more bonds to form one or more        optionally substituted carbocycles and/or heterocycles,        or R⁸+R^(8′) and/or R⁹+R^(9′) and/or R¹⁰+R^(10′) and/or        R¹¹+R^(11′) and/or R¹⁵+R^(15′) and/or R¹⁵″+R^(15′″) and/or        R¹⁶+R^(16′) and/or R²⁰+R^(20′) and/or R²¹+R^(21′) are        independently selected from ═O, ═S, ═NOR^(h1), ═C(R^(h1))R^(h2),        and ═NR^(h1), R^(h1) and R^(h2) being independently selected        from H and optionally substituted C₁₋₃ alkyl, two or more of R⁸,        R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵, R^(15′),        R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′), R²¹, R^(21′),        R²², and R²³ optionally being joined by one or more bonds to        form one or more optionally substituted carbocycles and/or        heterocycles;        R^(8b) and R^(9b) are independently selected and have the same        meaning as R⁸, except that they may not be joined with any other        substituent;        one of R⁴ and R^(4′) and one of R¹⁶ and R^(16′) may optionally        be joined by one or more bonds to form one or more optionally        substituted carbocycles and/or heterocycles;        one of R², R^(2′), R³, and R^(3′) and one of R⁵ and R^(5′) may        optionally be joined by one or more bonds to form one or more        optionally substituted carbocycles and/or heterocycles; and        a and b are independently selected from 0 and 1.

In a further aspect, this invention relates to a compound of formula(I′) or (II′):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein all substituents have the same meaning as described forcompounds of formulae (I) and (II). Compounds of formulae (I) and (II)are alleged to be converted to (I′) and (II′), respectively, in vivowith concomitant elimination of H—R¹.

Therefore, this invention relates to a compound of formula (I′) or(II′), said compound comprising a cyclopropyl group, which can be formedthrough rearrangement of and concomitant elimination of H—R¹ from acompound of formula (I) or (II). All embodiments for a compound offormula (I) or (II) or a moiety thereof also hold for a compound offormula (I′) or (II′) or a moiety thereof, unless the context dictatesotherwise.

In a more specific embodiment, this invention relates to a compound offormula (I) or (II) as described hereinabove, wherein

-   -   a) the DB moiety does not comprise a DA1, DA2, DA1′, or DA2′        moiety; and    -   b) ring B in DB1 is a heterocycle; and    -   c) if X³ in DB1 represents —X^(3a) and X^(3b)— and ring B is        aromatic, then two vicinal substituents on said ring B are        joined to form an optionally substituted carbocycle or        heterocycle fused to said ring B; and    -   d) if X³ in DB2 represents —X^(3a) and X^(3b)— and ring B is        aromatic, then two vicinal substituents on said ring B are        joined to form an optionally substituted heterocycle fused to        said ring B, an optionally substituted non-aromatic carbocycle        fused to said ring B, or a substituted aromatic carbocycle which        is fused to said ring B and to which at least one substituent is        attached that contains a hydroxy group, a primary amino group,        or a secondary amino group, the primary or secondary amine not        being a ring atom in an aromatic ring system nor being part of        an amide; and    -   e) if ring A in DB2 is a 6-membered aromatic ring, then        substituents on ring B are not joined to form a ring fused to        ring B; and    -   f) two vicinal substituents on ring A in DB8 are joined to form        an optionally substituted carbocycle or heterocycle fused to        said ring A to form a bicyclic moiety to which no further rings        are fused; and    -   g) ring A in DB9 together with any rings fused to said ring A        contains at least two ring heteroatoms.

In a further more specific embodiment, this invention relates to acompound of formula (I) or (II) as described hereinabove, wherein atleast one of the substituents R¹, R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′),R¹⁴, R^(14′), R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵,R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′), R²¹, R^(21′),R²², and R²³ contains a X¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴ moiety, wherein ff isselected from 1 to 1000 and each X¹⁴ is independently selected from

that is connected to the attachment site of said substituent either viaa direct bond or via a moiety, being part of said same substituent, thatdoes not comprise a disulfide, a hydrazone, a hydrazide, an ester, anatural amino acid, or a peptide containing at least one natural aminoacid, and wherein if ring B in DB1 is an all-carbon ring, X³ is O orNR¹⁵, X⁴ is CH, X³⁴ is C, there is only one X¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴moiety present in said compound of formula (I) or (II) and said moietyis part of R⁶, R⁷, R⁸, R¹⁰, or R¹⁵, then b=1 and ff is ≧5.

A compound of formula (I) or (II) or a conjugate thereof in which ff islarger than 1000 is encompassed by this invention.

In a further more specific embodiment, this invention relates to acompound of formula (I) or (II) as described hereinabove, wherein atleast one of the substituents R¹, R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′),R¹⁴, R^(14′), R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵,R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′), R²¹, R^(21′),R²², and R²³ contains a triazole moiety.

It should be understood that in this entire document, when referring toa compound of formula (I) or (II), this includes reference to a compoundof formula (I′) or (II′), respectively, unless structural parts of (I)and (II) not present in (I′) and (II′) are concerned or the contextdictates otherwise. Similarly, when referring to a structural part(fragment), linker-agent conjugate, or conjugate derived from a compoundof formula (I) or (II), this includes reference to a similar structuralpart (fragment), linker-agent conjugate, or conjugate derived from acompound of formula (I′) or (II′), respectively, unless structural partsof (I) and (II) not present in (I′) and (II′) are concerned or thecontext dictates otherwise.

It should also be understood that when reference is made to a compoundof formula (I) or (II) or a fragment, derivative, or conjugate thereofand the scope of R^(2′) or R¹² is specified, this specification onlyaffects a compound of formula (I) as R^(2′) and R¹² are absent in acompound of formula (II). Therefore, wherever it reads “R^(2′)” or “R¹²″in this document, one could read “R^(2′) (if present)” or “R¹² (ifpresent)”, respectively. This holds as well for (other) substituentsthat may be present or absent in compounds of formulae (I) and (II) andtheir fragments, linker-agent conjugates, and conjugates.

It should further be understood that this invention relates toenantiomerically pure and/or diastereomerically pure compounds offormulae (I) and (II) as well as to enantiomeric and/or diastereomericmixtures of compounds of formulae (I) and (II).

Considerations about substituent effects and the effects of linkers,DNA-alkylating units and DNA-binding units in compounds of formulae (I)and (II), their cyclopropyl-containing analogs, and their conjugates andlinker-agent conjugates given in this document are presented withoutconsenting to a specific mechanism of action for compounds of formulae(I) and (II), their cyclopropyl-containing analogs, and theirlinker-agent conjugates and conjugates.

Compounds of formula (I) and (II) can be considered to be built up of aDNA-binding unit (DB1-DB9) and a DNA-alkylating unit (DA1, DA2, DA1′, orDA2′), as indicated in the figures hereinabove.

The DNA-alkylating unit of compounds of formulae (I) and (II) isconsidered to contain the site of alkylation. Alkylation of DNA mayoccur through attack of DNA on the carbon bearing R¹ in a compound offormula (I) or (II) or on that same carbon in the cyclopropyl-containinganalog of said compound.

The DNA-binding unit of compounds of formulae (I) and (II) is consideredto assist in efficient binding of these compounds to DNA. It may becoupled to the DNA-alkylating moiety via, for instance, an amide bond.Therefore in one embodiment, X⁵ is O.

In one embodiment, this invention relates to a compound of formula (I).In another embodiment, this invention relates to a compound of formula(II).

R¹ in a compound of formula (I) or (II) is a leaving group.

In one embodiment, the leaving group R¹ is selected from halogen, azide(N₃), carboxylate [OC(O)R^(n)], carbonate [OC(O)OR^(n)], carbamate[OC(O)N(R^(n))R^(n1)], ⁺N(R^(n))(R^(n1)R^(n2), and S(O)₂R^(o), andOS(O)₂R^(o), wherein R^(n), R^(n1), R^(n2), and R^(o) are independentlyselected from H and optionally substituted C₁₋₁₀ alkyl, C₁₋₁₀heteroalkyl, C₅₋₁₀ aryl, or C₁₋₁₀ heteroaryl. An optional substituentmay be an oligoethylene glycol or a polyethylene glycol moiety. When theR¹ group comprises an oligoethylene glycol or polyethylene glycolmoiety, i.e., a X¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴ moiety, a compound of formula(I) or (II) or its conjugate may show improved physicochemical,biopharmaceutical, pharmacodynamic, and/or pharmacokinetic properties,which, as indicated hereinabove, may also be valid for the presence ofoligoethylene glycol or polyethylene glycol moieties at other positionsin a compound of formula (I) or (II). In addition, however, therelatively large size of the R¹ substituent may reduce non-specificalkylation of a compound of formula (I) or (II) or its conjugate.Furthermore, the R¹ group will be eliminated when the compound offormula (I) or (II) rearranges to a compound of formula (I′) or (II′).This means that the oligoethylene glycol or polyethylene glycol moietymay not have a negative effect on the cytotoxic potential of thecompound of formula (I) or (II).

In one embodiment, R¹ is selected from halogen and OS(O)₂R^(o). Inanother embodiment, the leaving group R¹ in a compound of formula (I) or(II) is a halogen. In another embodiment, R¹ is selected from chloro(Cl), bromo (Br), and iodo (I). In yet another embodiment, R¹ is chloro(Cl). In yet another embodiment, R¹ is bromo (Br). In yet anotherembodiment, R¹ is OS(O)₂R^(o). In yet another embodiment, R¹ isOS(O)₂R^(o) and R^(o) contains a X¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴ moiety. Inyet another embodiment, R¹ is selected from OS(O)₂CF₃, OS(O)₂C₆H₄CH₃,and OS(O)₂CH₃.

By varying the leaving group R¹, one may tune the alkylating activity ofthe seco agents and affect the transformation rate of a seco agent to acyclopropyl-containing agent of formula (I′) or (II′). If the leavingcapability of R¹ is too good, this may cause the seco agent to become anaspecific alkylating agent, which may decrease the cytotoxicity quotientand therapeutic index of conjugates of compounds of formulae (I) and(II) as the agent may for example be able to alkylate while still beingbound in the conjugate. On the other hand, if R¹ is too bad a leavinggroup, the seco agent may not close to form a cyclopropyl-containingagent, believed to be the active species, which may reduce itscytotoxicity and the cytotoxicity quotient. Therefore, in oneembodiment, the Swain-Scott parameter s of the alkylating site is largerthan 0.3. In other embodiments, the Swain-Scott parameter s is largerthan 0.5 or 0.7 or 1.0.

The size of R¹ may affect the non-DNA alkylation rate of a compound offormula (I) or (II) or a conjugate thereof. If R¹ is a relatively bulkygroup, aspecific alkylation may be reduced as the carbon bearing R¹ issomewhat shielded.

Another means to tune the alkylating activity of the seco agents andtheir cyclopropyl-containing derivatives may be to somewhat shield thecarbon to which the leaving group R¹ is attached or on whichnucleophilic attack can occur by choosing at least one of R², R^(2′),R³, R^(3′), R⁴, R^(4′), R⁵, R^(5′), R⁶, R^(6′), R¹², R¹⁶, and R^(16′)present to be other than hydrogen. Shielding of said carbon may reduceaspecific alkylation by compounds of formulae (I) and (II), theircyclopropyl-containing analogs, and their conjugates. Althoughintroduction of steric hindrance may also affect the DNA alkylationrate, it may be reasonable to assume that aspecific alkylation may beaffected relatively more than DNA alkylation as the latter occurspresumably after the agent is ideally positioned for nucleophilic attackbeing bound to the DNA minor groove. The carbon bearing R¹ in a compoundof formula (II), being a secondary carbon atom (when R² is H), isalready somewhat shielded in comparison to the carbon bearing R¹ in acompound of formula (I) when R² and R^(2′) are both H. In this respect,a compound of formula (II) may be compared to a compound of formula (I)in which R^(2′) is other than hydrogen. Further shielding may however beaccomplished by choosing one or more of R², R³, R^(3′), R⁴, R^(4′), R⁵,R^(5′), R⁶, R^(6′), R¹⁶, and R^(16′) present to be other than hydrogen.

In one embodiment, R² and R^(2′) are both hydrogen. In anotherembodiment, R^(2′) is hydrogen and R² is not hydrogen. In anotherembodiment, R² is selected from N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H,C(O)OH, halogen, R^(a), SR^(a), S(O)R^(a), S(O)₂R^(a), S(O)OR^(a),S(O)₂OR^(a), OS(O)R^(a), OS(O)₂R^(a), OS(O)OR^(a), OS(O)₂OR^(a), OR^(a),N(R^(a))R^(b), ⁺N(R^(a))(R^(b))R^(c), P(O)(OR^(a))(OR^(b)),OP(O)(OR^(a)(OR^(b)), SiR^(a)R^(b)R^(c), C(O)R^(a), C(O)OR^(a),C(O)N(R^(a))R^(b), OC(O)R^(a), OC(O)OR^(a), OC(O)N(R^(a))R^(b),N(R^(a))C(O)R^(b), N(R^(a))C(O)OR^(b), and N(R^(a))C(O)N(R^(b))R^(c),wherein R^(a), R^(b), and R^(c) are independently selected from H andoptionally substituted C₁₋₃ alkyl or C₁₋₃ heteroalkyl.

In one embodiment, R² is selected from optionally substituted C₁₋₃ alkyland C₁₋₃ heteroalkyl. In another embodiment, R² is optionallysubstituted C₁₋₃ alkyl. In another embodiment, R² is selected frommethyl, ethyl, propyl, and isopropyl. In another embodiment, R² ismethyl.

In yet another embodiment, R² and R^(2′) are both other than hydrogen.In one embodiment, both R² and R^(2′) are methyl.

Alternatively, or simultaneously, steric shielding of the carbon bearingR¹ may be introduced by choosing one or more of R³, R^(3′), R⁴, R^(4′),R¹², R¹⁶, and R^(16′) present to be other than hydrogen. In oneembodiment, R³, R^(3′), R⁴, and R^(4′) are each H. In anotherembodiment, R³ and R^(3′) are both H. In another embodiment, R⁴ andR^(4′) are both H. In another embodiment, one of R³ and R^(3′) is C₁₋₃alkyl while the other is H. In another embodiment, one of R⁴ and R^(4′)is C₁₋₃ alkyl while the other is H. In another embodiment, one of R³ andR^(3′) is C₁₋₃ alkyl and one of R⁴ and R^(4′) is C₁₋₃ alkyl while theothers are H. In another embodiment, both R³ and R^(3′) areindependently C₁₋₃ alkyl. In another embodiment, both R⁴ and R^(4′) areindependently C₁₋₃ alkyl. In another embodiment, one of R³, R^(3′), R⁴,and R^(4′) is methyl. In another embodiment, one of R⁴ and R^(4′) ismethyl. In yet another embodiment, both R⁴ and R^(4′) are methyl. In yetother embodiments, one or both of R⁴ and R^(4′) are fluoro.

In one embodiment, R¹² is H. In another embodiment, R¹² is C₁₋₃ alkyl.In yet other embodiments, R¹² is methyl or ethyl. In yet anotherembodiment, R¹² equals C(R^(2′))(R²)R¹, which means that the carbonbearing R¹² bears two identical groups.

In another embodiment, R¹⁶ and R^(16′) are both H. In anotherembodiment, R¹⁶ is H. In other embodiments, R¹⁶ is fluoro (F) or methylor ethyl.

The alkylating activity of a compound of formula (I) or (II) or itscyclopropyl-containing analog may also be affected by the nature of X¹.The nature of X¹ may affect the rate at which and the conditions underwhich the seco agents ring close to the cyclopropyl analogs and/or therate at which the cyclopropyl ring is opened by nucleophilic attack (byDNA), and thus affect the alkylation behavior. In one embodiment, X¹ isO. In another embodiment, X¹ is NR¹³.

The substituents R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′), and X² as well asthe size of the ring connected to the left-hand side of the ring bearingX¹ may for example, each independently or two or more taken together,affect the pharmacological properties of the agent, e.g., affect thewater solubility, affect the aggregation behavior, affect the DNAalkylation process, and/or affect the DNA binding strength. Furthermore,especially R⁵ and R^(5′), and to some degree R⁶ and R^(6′) as well, mayalso affect the degree of shielding of the carbon on which nucleophilicattack should occur.

R⁵ and R^(5′) may both be H, or R⁵ may be H while R^(5′) is absent. Inanother embodiment, at least one of R⁵ and R^(5′) is not hydrogen norabsent. In another embodiment, R⁵ is not hydrogen.

In one embodiment, R⁵ is selected from OH, SH, NH₂, N₃, NO₂, NO, CF₃,CN, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(e2), SR^(e2), S(O)R^(e2),S(O)₂R^(e2), S(O)OR^(e2), S(O)₂OR^(e2), OS(O)R^(e2), OS(O)₂R^(e2),OS(O)OR^(e2), OS(O)₂OR^(e2), OR^(e2), NHR^(e2), N(R^(e2))R^(f2),N(R^(e2))(R^(f2))R^(g2), P(O)(OR^(e2))(OR^(f2)),OP(O)(OR^(e2))(OR^(f2)), SiR^(e2)R^(f2)R^(g2), C(O)R^(e2), C(O)OR^(e2),C(O)N(R^(e2))R^(f2), OC(O)R^(e2), OC(O)OR^(e2), OC(O)N(R^(e2))R^(f2),N(R^(e2))C(O)R^(f2), N(R^(e2))C(O)OR^(f2), andN(R^(e2))C(O)N(R^(f2))R^(g2), wherein R^(e2), R^(f2), and R^(g2) areindependently selected from H and optionally substituted C₁₋₃ alkyl,C₁₋₃ heteroalkyl, C₃ cycloalkyl, or C₁₋₃ heterocycloalkyl, two or moreof R^(e2), R^(f2), and R^(g2) optionally being joined by one or morebonds to form one or more optionally substituted carbocycles and/orheterocycles.

In another embodiment, R⁵ is selected from OH, SH, NH₂, N₃, NO₂, NO,CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(e2), SR^(e2), S(O)R^(e2),S(O)₂R^(e2), S(O)OR^(e2), S(O)₂OR^(e2), OS(O)R^(e2), OS(O)₂R^(e2),OS(O)OR^(e2), OS(O)₂OR^(e2), OR^(e2), NHR^(e2), N(R^(e2))R^(f2),N(R^(e2))(R^(f2))R^(g2), P(O)(OR^(e2))(OR^(f2)),OP(O)(OR^(e2))(OR^(f2)), SiR^(e2)R^(f2)R^(g2), C(O)R^(e2), C(O)OR^(e2),C(O)N(R^(e2))R^(f2), OC(O)R^(e2), OC(O)OR^(e2), OC(O)N(R^(e2))R^(f2),N(R^(e2))C(O)R^(f2), N(R^(e2))C(O)OR^(f2), andN(R^(e2))C(O)N(R^(f2))R^(g2), wherein R^(e2), R^(f2), and R^(g2) areindependently selected from H and optionally substituted C₁₋₃ alkyl,C₁₋₃ heteroalkyl, C₃ cycloalkyl, or C₁₋₃ heterocycloalkyl, two or moreof R^(e2), R^(f2), and R^(g2) optionally being joined by one or morebonds to form one or more optionally substituted carbocycles and/orheterocycles, provided that R^(e2) is not H when R⁵ is R^(e2).

In another embodiment, R⁵ is selected from nitro, halogen, amino, cyano,hydroxy, and optionally substituted C₁₋₃ alkylamino, di(C₁₋₃alkyl)amino, C₁₋₃ alkylcarbonylamino, C₁₋₃ alkoxycarbonylamino, C₁₋₃alkylaminocarbonylamino, C₁₋₃ alkyloxy, C₁₋₃ alkylcarbonyloxy, C₁₋₃alkoxycarbonyloxy, C₁₋₃ alkylaminocarbonyloxy, or C₁₋₃ alkyl. In yetanother embodiment, R⁵ is optionally substituted linear C₁₋₃ alkyl. Inanother embodiment, R⁵ is unsubstituted linear C₁₋₃ alkyl. In anotherembodiment, R⁵ is selected from methyl, ethyl, propyl, isopropyl, nitro,CF₃, F, Cl, Br, cyano, methoxy, ethoxy, propoxy, isopropoxy, amino(NH₂), methylamino, formyl, hydroxymethyl, and dimethylamino. In anotherembodiment, R⁵ is methyl, ethyl, methoxy, or ethoxy. In anotherembodiment, R⁵ is methyl. In other embodiments, R⁵ is ethyl or methoxyor ethoxy.

R⁶ and R^(6′) may both be hydrogen, or R⁶ may be hydrogen while R^(6′)is absent. In another embodiment, at least one of R⁶ and R^(6′) is nothydrogen nor absent. In another embodiment, R⁶ is not hydrogen.

R⁵ and R⁶ may be joined to form, together with the two carbon atoms towhich they are attached, an optionally substituted 5- or 6-memberedring. This ring may for example be a dihydropyrrole, dihydrofuran,cyclopentene, 1,3-dioxolene, pyrrolidine, tetrahydrofuran, cyclopentane,or 1,3-dioxo lane moiety.

The substituents R¹⁶ and R^(16′) may affect the degree of shielding ofthe carbon on which nucleophilic attack can occur as well. In oneembodiment X⁴ is CR¹⁶. In a further embodiment, R¹⁶ is hydrogen. In yetanother embodiment, R¹⁶ is C₁₋₃ alkyl or C₁₋₃ heteroalkyl. In anotherembodiment, R¹⁶ is methyl or ethyl. In yet another embodiment, R¹⁶ ismethyl. In yet another embodiment, R¹⁶ is fluoro.

R¹⁴ and R^(14′) may affect the degree of shielding of X¹, or when acompound of formula (I) or (II) is part of a conjugate or linker-agentconjugate in which the compound of formula (I) or (II) is connected viaX¹, they may affect the degree of shielding of the linkage between thecompound of formula (I) or (II) and the promoiety. To increase thestability of this linkage, R¹⁴ may be selected to be other thanhydrogen. In one embodiment, R¹⁴ is hydrogen. In another embodiment, R¹⁴is methyl. In yet other embodiments, R¹⁴ is chloro or ethyl orisopropyl. In yet another embodiment, R⁵ and R¹⁴ are the same and nothydrogen. For example, both R⁵ and R¹⁴ may be methyl.

In one embodiment, R², R^(2′), R³, R^(3′), R⁴, R^(4′), R⁵, R^(5′), R⁶,R^(6′), R¹², R¹⁶, and R^(16′) present are each hydrogen. In anotherembodiment R², R^(2′), R³, R^(3′), R⁴, R^(4′), R^(5′), R⁶, R^(6′), R¹²,R¹⁶, and R^(16′) present are each hydrogen. In yet another embodiment,R², R^(2′), R³, R^(3′), R⁴, R^(4′), R⁵, R^(5′), R⁶, R^(6′), R¹², R¹⁴,R^(14′), R¹⁶, R^(16′), and R¹⁹ present are each hydrogen. In yet anotherembodiment, R², R^(2′), R³, R^(3′), R⁴, R^(4′), R⁵, R^(5′), R⁶, R^(6′),R¹², R¹⁴, R^(14′), R¹⁶, and R^(16′), and R¹⁹ present are each hydrogen.

Although the alkylation rate and efficiency of compounds of formulae (I)and (II) may optionally be tuned in several ways, in one aspect of thisinvention, this may be achieved by introducing steric shielding choosingfor a compound of formula (I) one or more of R², R^(2′), R³, R^(3′), R⁴,R^(4′), R⁵, R^(5′), R⁶, R^(6′), R¹², R¹⁶, and R^(16′) present to beother than hydrogen and for a compound of formula (II) one or more ofR², R³, R^(3′), R⁴, R^(4′), R⁵, R^(5′), R⁶, R^(6′), R¹⁶, and R^(16′)present to be other than hydrogen. Substituents should not cause toomuch steric hindrance, however, especially when more than one of thesesubstituents is other than hydrogen, as this might adversely affect DNAalkylation. Furthermore, it may provide for less efficient binding inthe DNA minor groove and may pose synthetic difficulties.

In one aspect of this invention, at least one of R¹, R⁵, R^(5′), R⁶,R^(6′), R⁷, R^(7′), R¹⁴, R^(14′), R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′),R¹¹, R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰,R^(20′), R²¹, R^(21′), R²², and R²³ contains aX¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴ moiety, wherein ff is selected from 1 to 1000and each X¹⁴ is independently selected from

This moiety must be connected to the core of the DNA-alkylating moietyor DNA-binding moiety via a direct bond or via a linking unit that ispart of said same R group and that does not comprise a disulfide, ahydrazone, a hydrazide, an ester, a natural amino acid, or a peptidecontaining at least one natural amino acid. Said linking unit shouldpreferably be cleaved less than 20%, more preferably less than 10%, andmost preferably less than 5% in 24 hours upon administration of acompound of formula (I) or (II) in vivo.

The X¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴ moiety may for example be selected to be

wherein ff is selected from 1 to 1000. In more specific embodiments, ffis selected from 1 to 100 or from 1 to 10. In other embodiments, ff isselected to be 1 or 2 or 3 or 4. In another embodiment, ff is 3 or 4.

The oligoethylene glycol or polyethylene glycol moiety or derivativethereof is connected via a linking unit to the core structure of acompound of formula (I) or (II). Such a linking unit may be a singlebond, in which case the oligoethylene glycol or polyethylene glycol orderivative thereof is connected to the core structure via for example anamine, ether, or sulfide bond. Alternatively, the oligoethylene glycolor polyethylene glycol moiety or derivative thereof may be connected tothe core structure via for example a carbamate, a carbonate, an amide,an alkyl, a heteroalkyl, an aryl, or a heteroaryl moiety, or acombination of any of these. In one embodiment, at least one of R¹, R⁵,R^(5′), R⁶, R^(6′), R⁷, R^(7′), R¹⁴, R^(14′), R⁸, R^(8′), R⁹, R^(9′),R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″), R¹⁶,R^(16′), R²⁰, R^(20′), R²¹, R^(21′), R²², and R²³ is selected from

wherein hh is selected from 1 to 1000, X¹⁵ is selected from S and NR³²,each X¹⁶ is independently selected from O, S, and NR³⁴, R³⁰ isindependently selected from H and optionally substituted C₁₋₁₀ alkyl,C₁₋₁₀ heteroalkyl, C₃₋₁₀ cycloalkyl, C₁₋₁₀ heterocycloalkyl, C₅₋₁₀ aryl,or C₁₋₁₀ heteroaryl, R³², R³³, and R³⁴ are independently selected from Hand C₁₋₃ alkyl, and R³¹ has the same meaning as defined for R⁷. R³⁰ mayfor example be selected from H, methyl, ethyl, methoxymethyl,p-aminobenzoyl, and p-aminoanilinocarbonyl.

In a further embodiment R¹, R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′), R¹⁴,R^(14′), R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵,R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′), R²¹, R^(21′),R²², and R²³ is selected from

In another embodiment, R¹ is selected from

In one embodiment R¹, R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′), R¹⁴, R^(14′),R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵, R^(15′),R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′), R²¹, R^(21′), R²², andR²³ contains a X¹⁴ (CH₂CH₂O)_(ff)CH₂CH₂X¹⁴ moiety. In anotherembodiment, at least one of R⁵, R⁶, R⁷, and R¹⁴ contains aX¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴ moiety. In yet another embodiment, at leastone of R⁶ and R⁷ contains a X¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴ moiety. In yetanother embodiment, at least one of R⁸, R^(8′), R⁹, R^(9′), R¹⁰,R^(10′), R¹¹, R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′),R²⁰, R^(20′), R²¹, R^(21′), R²², and R²³ contains aX¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴ moiety. In yet another embodiment, at leastone of R⁸, R⁹, R¹⁰, R¹¹, R²⁰, R²¹, and R²² contains aX¹⁴(CH₂CH₂O)_(f)/CH₂CH₂X¹⁴ moiety. In yet another embodiment, at leastone of R⁸ and R⁹ contains a X¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴ moiety. In yetanother embodiment, at least R¹ contains a X¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴moiety.

A compound of formula (I) or (II) may also contain 2 or moreX¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴ moieties. In one embodiment, a compound offormula (I) or (II) contains 2 X¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴ moieties. Inanother embodiment, a compound of formula (I) or (II) contains 2X¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴ moieties that are part of 2 separate Rgroups. It may be beneficial to put the two or moreX¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴ moieties at distant positions in the compoundof formula (I) or (II) as this may shield the relatively hydrophobiccore more efficiently.

Compounds of formulae (I) and (II) may contain one or more oligoethyleneglycol or polyethylene glycol moieties or derivatives thereof. Such amoiety may improve the water solubility and aggregation behavior of acompound of formula (I) or (II) and may cause increased activity againstmultidrug-resistant targets due to increased polarity. If a compound offormula (I) or (II) with such a moiety is incorporated in a conjugate,it may be that the oligoethylene glycol or polyethylene glycol moiety islocated in between the promoiety and the remainder of the compound offormula (I) or (II) or that it is located at a position somewhatopposite to the attachment site of the promoiety, thus placing theremainder of the compound of formula (I) or (II) in between thepromoiety and the oligoethylene glycol or polyethylene glycol moiety.The latter may be more beneficial for the water solubility of theconjugates. Improved water solubility of compounds of formulae (I) and(II) and their conjugates may lead to improved yields and purity of theconjugates during synthesis, for example due to reduced aggregateformation. Furthermore, a reduced tendency for aggregation and a higherpurity of the conjugate may for example lead to fewer side effects afteradministration of the conjugate. In addition, the presence of one ormore oligoethylene glycol and/or polyethylene glycol moieties in aconjugate may reduce excretion of the conjugate via the kidneys orliver, which increases the circulation time in the body.

In another aspect of this invention, compounds of formula (I) and (II)may contain one or more triazole rings. Incorporation of a1,2,3-triazole ring may provide for a synthetic advantage as the twomoieties that eventually may become attached to the 1,2,3-triazole ringmay be attached to each other via said triazole ring using a mild andefficient cycloaddition reaction between an alkyne and azide moiety.Because the conditions for this cycloaddition reaction are very mild andare compatible with almost all functional groups, the reaction can beperformed in one of the last steps of the synthetic route towards acompound of formula (I) or (II), its linker-agent conjugate, orconjugate, thus allowing for easy generation of series of compounds offormula (I) and (II) and their conjugates for SAR (structure-activityrelationship) studies.

Preferably, the triazole moiety is located in such a way within theDNA-alkylating unit or DNA-binding unit that it can contribute to thebinding of the compound to DNA. Additional DNA-binding moieties, such asindole or benzofuran moieties, that are connected to the DNA-binding orDNA-alkylating unit may increase the potency of the compound, allegedlythrough enhanced DNA binding. These additional aromatic moieties mayhowever have a detrimental effect on pharmacological properties, such aswater solubility. A triazole, being an aromatic group, may also enhancebinding to DNA and thus increase cytotoxic potency of the compound, butas it is more polar than other aromatic moieties such as a phenyl ring,negative effects on pharmacological properties may be less pronounced.

In one embodiment, this invention relates to a compound of formula (I)or (II) wherein at least one of R¹, R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′),R¹⁴, R^(14′), R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵,R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′), R²¹, R^(21′),R²², and R²³ contains a triazole moiety.

In another embodiment, at least one of R⁸, R^(8′), R⁹, R^(9′), R¹⁰,R^(10′), R¹¹, R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′),R²⁰, R^(20′), R²¹, R^(21′), R²², and R²³ contains a triazole moiety. Inanother embodiment, at least one of R⁸, R⁹, and R¹⁰ contains a triazolemoiety. In another embodiment, at least one of R⁸ and R⁹ contains atriazole moiety. In yet another embodiment, at least R⁸ contains atriazole moiety.

In another embodiment, at least one of R⁵, R^(5′), R⁶, R^(6′), R⁷,R^(7′), R¹⁴, and R^(14′) contains a triazole moiety. In anotherembodiment, at least one of R⁶, R^(6′), R⁷, and R^(7′) contains atriazole moiety. In yet another embodiment, R¹ contains a triazolemoiety.

For an optimum DNA-binding effect, the triazole moiety may be connectedvia a linker that keeps the triazole moiety in conjugation with or inclose proximity to the core of the DNA-binding or DNA-alkylating unit.The linker may for example be a single bond, —N(R³⁵)C(O)—, —C(O)N(R³⁵)—,—C(O)—, —C(R³⁵)(R³⁶)—, —C(R³⁵)═C(R³⁶)—, —O—, —S—, or —N(R³⁵)—, whereinR³⁵ and R³⁶ are selected from H and optionally substituted C₁₋₄ alkyl orC₁₋₄ heteroalkyl, or be any other optionally substituted small linkerthat does not have more than 4 connecting atoms (e.g., the —N(R²⁰)C(O)—moiety has two connecting atoms: N and C) in between the core of theDNA-binding unit or DNA-alkylating unit and the triazole ring.

The triazole ring may be a 1,2,3-triazole or a 1,2,4-triazole. In oneembodiment, the triazole ring is a 1,2,3-triazole. In anotherembodiment, the triazole is a 1,2,4-triazole. A 1,2,3-triazole ring maybe 4,5-, 1,5-, or 1,4-disubstituted. If the 1,2,3-triazole ring is1,4-substituted, this means that the substituent that contains the1,2,3-triazole ring has an extended form. If the 1,2,3-triazole ring is4,5- or 1,5-substituted, the 1,2,3-triazole ring in fact forms a kind ofturn and puts the two substituents on the triazole in close proximity toeach other. The triazole ring may also be located at the end of thesubstituent, in which case the triazole ring is only monosubstituted.Substitution may in this case occur at N-1 or C-4. A 1,2,4-triazole maybe 1,3-, 1,5-, or 3,5-disubstituted. A substituent that contains a 1,3-or 3,5-disubstituted 1,2,4-triazole has an extended form, whereas in a1,5-disubstituted 1,2,4-triazole both substituents on the triazole arein close proximity to each other. The triazole ring may also betrisubstituted.

In one aspect, at least one of R¹, R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′),R¹⁴, R^(14′), R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵,R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′), R²¹, R^(21′),R²², and R²³ in a compound of formula (I) or (II) is

wherein X¹⁸ and X¹⁹ are selected from O, S, NR²⁵, H₂, and C(R²⁵)R²⁶,wherein R²⁵ and R²⁶ are selected from H and optionally substituted C₁₋₃alkyl or C₁₋₃ heteroalkyl, and R²⁴ has the same meaning as R⁸ and isindependently selected.

R²⁴ may for example be selected from H and

wherein jj, jj′, jj″, and jj′″ are independently selected from 0 to 8,X⁷⁴ is selected from

each tt, tt′, and tt″ is independently selected from 0 and 1, each X²¹and X²² is independently selected from O, S, NR⁶⁷, H₂, and C(R⁶⁷)R⁶⁸,wherein R⁶⁷ and R⁶⁸ are independently selected from H and optionallysubstituted C₁₋₃ alkyl or C₁₋₃ heteroalkyl, and R⁶⁶ is selected from H,COOH, CO₂Me, OH, OMe, NR⁶⁹R⁷⁰, NR⁶⁹C(O)CH₃, SH, SMe,

wherein X²³ is selected from halide, hydroxy, OC(O)R^(bb), andOC(O)OR^(bb), or C(O)—X²³ is an active ester, X²⁴ is selected fromhalide, mesyloxy, triflyloxy, and tosyloxy, R^(bb) is selected fromoptionally substituted C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, C₃₋₁₀ cycloalkyl,C₁₋₁₀ heterocycloalkyl, C₅₋₁₀ aryl, and C₁₋₁₀ heteroaryl, and R⁶⁹, R⁷⁰,and R⁷¹ are independently selected from methyl and H.

In other embodiments, at least one of R⁵, R⁶, R⁷, and R¹⁴, or at leastone of R⁸, R⁹, R¹¹ and R″, or at least one of R⁶ and R⁷, or at least oneof R⁸ and R⁹, or at least R⁸, or at least R⁶, or at least R⁷ in acompound of formula (I) or (II) is

wherein R²⁴, X¹⁸, and X¹⁹ are as defined hereinabove.

In some embodiments, at least one of R¹, R⁵, R^(5′), R⁶, R^(6′), R⁷,R^(7′), R¹⁴, R^(14′), R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹,R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′),R²¹, R^(21′), R²², and R²³, or at least one of R⁸, R⁹, R¹⁰, and R¹¹, orat least one of R⁸ and R⁹, or at least R⁸, or at least one of R⁵, R⁶,R⁷, and R¹⁴, or at least one of R⁶ and R⁷ in a compound of formula (I)or (II) is selected from

wherein R³⁷, R³⁸, R³⁹, and R⁴⁰ are independently selected from H andmethyl.

In other embodiments, at least one of R¹, R⁵, R^(5′), R⁶, R^(6′), R⁷,R^(7′), R¹⁴, R^(14′), R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹,R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′),R²¹, R^(21′), R²², and R²³, or at least one of R⁸, R⁹, R¹⁰, and R¹¹, orat least one of R⁸ and R⁹, or at least R⁸, or at least one of R⁵, R⁶,R⁷, and R¹⁴, or at least one of R⁶ and R⁷ in a compound of formula (I)or (II) is selected from

R³⁸, R³⁹, R⁴⁰ are independently selected from H and methyl.

In other embodiments R¹, R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′), R¹⁴,R^(14′), R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵,R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′), R²¹, R^(21′),R²², and R²³, or at least one of R⁸, R⁹, R¹⁰, and R¹¹, or at least oneof R⁸ and R⁹, or at least R⁸, or at least one of R⁵, R⁶, R⁷, and R¹⁴, orat least one of R⁶ and R⁷ in a compound of formula (I) or (II) isselected from

wherein R³⁸, R³⁹, and R⁴⁰ are independently selected from H and methyl.

In other embodiments R¹, R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′), R¹⁴,R^(14′), R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵,R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′), R²¹, R^(21′),R²², and R²³, or at least one of R⁸, R⁹, R¹⁰, and R¹¹, or at least oneof R⁸ and R⁹, or at least R⁸, or at least one of R⁵, R⁶, R⁷, and R¹⁴, orat least one of R⁶ and R⁷ in a compound of formula (I) or (II) isselected from

wherein R³⁸, R³⁹, and R⁴⁰ are independently selected from H and methyl.

In one aspect, compounds of formulae (I) and (II) are represented bycompounds of formulae (Ib) and (IIb), respectively:

In one embodiment, X² in (Ib) or (IIb) is N.

In another embodiment, X² in (Ib) or (IIb) is CR¹⁴.

In a further embodiment, X² in (Ib) is CR¹⁴ and a is 0.

In another embodiment, X² in (Ib) or (IIb) is CH.

In yet another embodiment, R⁵ in (Ib) or (IIb) is selected from nitro,halogen, amino, cyano, hydroxy, and optionally substituted C₁₋₃alkylamino, di(C₁₋₃ alkyl)amino, C₁₋₃ alkylcarbonylamino, C₁₋₃alkoxycarbonylamino, C₁₋₃ alkylaminocarbonylamino, C₁₋₃ alkyloxy, C₁₋₃alkylcarbonyloxy, C₁₋₃ alkylaminocarbonyloxy, or C₁₋₃ alkyl. In yetanother embodiment, R⁵ in (Ib) or (IIb) is optionally substituted linearC₁₋₃ alkyl. In another embodiment, R⁵ in (Ib) or (IIb) is unsubstitutedlinear C₁₋₃ alkyl. In another embodiment, R⁵ in (Ib) or (IIb) is methyl.In other embodiments, R⁵ in (Ib) or (IIb) is ethyl or methoxy or ethoxy.

In yet another aspect, compounds of formulae (I) and (II) arerepresented by compounds of formulae (Ic) and (IIc), respectively:

In one embodiment, X² in (Ic) or (IIc) is NH.

In yet another aspect, compounds of formulae (I) and (II) arerepresented by compounds of formulae (Id) and (IId), respectively:

In one embodiment, X² in (Id) or (Hd) is NH.

In another embodiment, compounds of formulae (I) and (II) arerepresented by (Ia) and (IIa), respectively:

DA1-DB  (Ia)

DA2-DB  (IIa)

wherein DA1 is

or an isomer or a mixture of isomers thereof.

In other embodiments, compounds of formulae (I) and (II) are representedby (Ia) and (IIa), respectively:

DA1-DB  (Ia)

DA2-DB  (IIa)

wherein DA1 is

or an isomer of one of these, or a mixture of isomers.

In other embodiments, compounds of formulae (I) and (II) are representedby (Ia) and (IIa), respectively:

DA1-DB  (Ia)

DA2-DB  (IIa)

wherein DA1 is

or an isomer of one of these, or a mixture of isomers.

In yet other embodiments, compounds of formulae (I) and (II) arerepresented by (Ia) and (IIa), respectively:

DA1-DB  (Ia)

DA2-DB  (IIa)

wherein DA1 is

wherein R⁵⁴ is selected from H and optionally substituted C₁₋₃ alkyl(e.g., methyl or trifluoromethyl), R⁵⁵ is selected from H, methyl,ethyl, and methoxy, X²⁵ and X²⁶ are independently selected from O, S,CH₂, and NR⁵¹, and R⁵¹, R⁵², and R⁵³ are independently selected from H,C₁₋₃ alkyl and

wherein ii, ii′, ii″ and ii′″ are independently selected from 0 to 8,X⁷⁴ is selected from

each ss, ss′, and ss″ is independently selected from 0 and 1, each X²⁵and X²⁶ is independently selected from O, S, NR⁵⁶, H₂, and C(R⁵⁶)R⁵⁷,wherein R⁵⁶ and R⁵⁷ are independently selected from H and optionallysubstituted C₁₋₃ alkyl or C₁₋₃ heteroalkyl, and R⁵⁸ is selected from H,COOH, CO₂Me, OH, OMe, NR⁵⁹R⁶⁰NR⁵⁹C(O)CH₃, SH, SMe,

wherein X²⁷ is selected from halide, hydroxy, OC(O)R^(aa), andOC(O)OR^(aa), or C(O)—X²³ is an active ester, X²⁴ is selected fromhalide, mesyloxy, triflyloxy, and tosyloxy, R^(aa) is selected fromoptionally substituted C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, C₃₋₁₀ cycloalkyl,C₁₋₁₀ heterocycloalkyl, C₅₋₁₀ aryl, and C₁₋₁₀ heteroaryl, and R⁵⁹, R⁶⁹,and R⁶¹ are independently selected from methyl and H, or an isomer ofone of these, or a mixture of isomers.

In another embodiment, a compound of formula (I) or (II) is

or an isomer thereof, or a mixture of isomers.

In another embodiment, a compound of formula (I) or (II) is

or an isomer thereof, or a mixture of isomers.

In other embodiments, a compound of formula (I) or (II) is

or an isomer of one of these, or a mixture of isomers.

In one embodiment, in a compound of formula (I) or (II), b=1. In anotherembodiment, b=0. In another embodiment, a=0. In yet another embodiment,a=1. In yet another embodiment, a=0 and b=1.

Increased water solubility of a compound of formula (I) or (II) may notonly be achieved through the introduction of water-soluble or polargroups, such as a triazole group or an oligoethylene glycol orpolyethylene glycol moiety or a combination thereof, but may also beachieved through substitution of carbon ring atoms by heteroatoms, forexample in the DNA-binding unit. Improved water solubility of compoundsof formulae (I) and (II) and their conjugates may lead to improvedyields and purity of the conjugates during synthesis, for example due toreduced aggregate formation. Furthermore, a reduced tendency foraggregation and a higher purity of the conjugate may for example lead tofewer side effects after administration of the conjugate.

Increased metabolic degradation, e.g., in the liver, may for example beachieved through the introduction of groups in the DNA-binding unitsthat can be oxidized with relative ease, for example acetylene andalkene moieties. Oxidation of toxic compounds is one of the mechanismsby which a mammal may detoxify such compounds. If compounds of thisinvention are taken up in the liver, efficient detoxification may forexample circumvent liver toxicity as a side effect.

The stability of the linkage between the DNA-binding unit andDNA-alkylating unit, for example in the circulation, may be tuned bymodifications in the DNA-binding unit. It may be favorable to have theDNA-alkylating unit and DNA-binding unit linked via an essentiallynon-cleavable bond. Especially when the compound of formula (I) or (II)is administered as the active ingredient, stability in the circulationmay be desired. However, when a compound of formula (I) or (II) isadministered as a part of a conjugate, it may be favorable to have abond between the DNA-alkylating unit and the DNA-binding unit thatbecomes less stable in the circulation as soon as the compound offormula (I) or (II) is prematurely released from the conjugate. This mayreduce toxic side effects as a consequence of premature release of theagent. The DNA-binding units of this invention may cause a compound offormula (I) or (II) or its conjugate or linker-agent conjugate to have amore stable linkage between DNA-binding and DNA-alkylating unit in thecirculation than a compound of formula (I′) or (II′), which may beformed from a compound of formula (I) or (II) as soon as X¹ isunprotected.

Extension of the π-conjugated system in the DNA-binding moiety mayincrease the binding affinity of the DNA binder for DNA. The π systemmay be extended by the introduction of additional aromatic rings and/orconjugated double and/or triple bonds.

Promoieties may be connected to the DNA-binding units if a suitablefunctional group is present. This may for example be a hydroxyl group ora primary or secondary amino group. Coupling of a promoiety to theDNA-binding unit in addition to or instead of to the alkylating unit,e.g., to X¹, may provide advantages. For example, the presence of twopromoieties may increase target-selective delivery and/or activationand/or reduce the amount of free agent in non-targeted areas, therebyreducing side effects and increasing the therapeutic index.

The DNA-binding unit DB in a compound of formula (I) or (II) is selectedfrom structures DB1-DB9:

In one embodiment, the DNA-binding unit comprises at least two aromaticrings of which at least one contains at least one ring atom that is aheteroatom or the DNA-binding unit comprises at least a bicyclicaromatic system in which at least one ring atom is a heteroatom. Inanother embodiment, the DNA-binding unit comprises at least two aromaticrings and both contain at least one ring atom that is a heteroatom orthe DNA-binding unit comprises at least a bicyclic aromatic system inwhich at least two ring atoms are a heteroatom.

In one aspect of this invention, a compound of formula (I) or (II) has aDNA-binding unit of formula DB1. This moiety comprises structures thatat least contain a 6-membered ring B that is connected to theDNA-alkylating unit via a fused 5- or 6-membered ring A or vinyl group.The optional heteroatom in said ring B may provide for improved watersolubility with respect to DNA binder analogs having an all-carbon ring.In one embodiment, ring B in unit DB1 contains a heteroatom.

Preferably, ring B is aromatic. It may for example be a phenyl,pyridine, pyrimidine, pyridazine, pyrazine, 1,3,5-triazine,1,2,3,5-tetrazine, 1,2,3,4-tetrazine, pentazine, phosphinine,1,3-diphosphinine, or 1,3-azaphosphinine moiety. Alternatively, thisring may be non-aromatic and either be unsaturated or completelysaturated.

A compound of formula (I) or (II) wherein ring B is connected to theDNA-alkylating unit via a vinyl group may contain a handle that allowsfor detoxification by means of for example oxidation or hydration of thedouble bond.

The moiety DB1 may for example be

Moiety DB1 may for example also be

In another embodiment, the moiety DB1 may be

In another embodiment, the moiety DB1 may be

In another embodiment, the moiety DB1 may for example be

wherein R^(9a) has the same meaning as defined for R⁹ and isindependently selected.

In a further embodiment, the moiety DB1 may be selected from

In one embodiment, the moiety DB1 is

In another embodiment, the moiety DB1 is

In yet another embodiment, the moiety DB1 is

The moiety DB1 may for example also be

In the exemplary structures of DB1, R⁸, R^(8′), R⁹, R^(9′), R^(9a), R¹⁰,R^(10′), R¹¹, R^(11′), R¹⁵, R¹⁶, and R²¹ may for example eachindependently be selected to be H, be or contain another moiety selectedfrom structures DB1-DB9 or a derivative thereof, or be

wherein R⁶², R⁶³, R⁶⁴, and R⁶⁵ are independently selected from H, C₁₋₃alkyl, and

wherein jj, jj′, jj″, and jj′″ are independently selected from 0 to 8,X⁷⁴ is selected from

each tt, tt′, and tt″ is independently selected from 0 and 1, each X²¹and X²² is independently selected from O, S, NR⁶⁷, H₂, and C(R⁶⁷)R⁶⁸,wherein R⁶⁷ and R⁶⁸ are independently selected from H and optionallysubstituted C₁₋₃ alkyl or C₁₋₃ heteroalkyl, and R⁶⁶ is selected from H,COOH, CO₂Me, OH, OMe, NR⁶⁹R⁷⁰, NR⁶⁹C(O)CH₃, SH, SMe,

wherein X²³ is selected from halide, hydroxy, OC(O)R^(bb), andOC(O)OR^(bb), or C(O)—X²³ is an active ester, X²⁴ is selected fromhalide, mesyloxy, triflyloxy, and tosyloxy, Rbb is selected fromoptionally substituted C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, C₃₋₁₀ cycloalkyl,C₁₋₁₀ heterocycloalkyl, C₅₋₁₀ aryl, and C₁₋₁₀ heteroaryl, and R⁶⁹, R⁷⁰,and R⁷¹ are independently selected from methyl and H.

In a further embodiment, the moiety DB1 may for example be

In a further embodiment, the moiety DB1 may be selected from

In a further embodiment, the moiety DB1 may be selected from

In a further embodiment, the moiety DB1 may be selected from

In another embodiment, the moiety DB1 may for example be

In another aspect of this invention, a compound of formula (I) or (II)has a DNA-binding unit of formula DB2. This moiety comprises structuresthat at least contain a 5-membered ring B that is connected to theDNA-alkylating unit via a fused 5- or 6-membered ring A or a vinylgroup. Especially in the latter case, ring B may be fused to anotherheterocyclic or carbocyclic aromatic or non-aromatic ring in order tohave an improved DNA-binding affinity. For reasons of increased watersolubility, the fused ring may be a heterocycle, or a carbocyclesubstituted with relatively polar groups that at the same time mayprovide handles for coupling to promoieties. A DNA binder in which threeor more rings are fused together to form an aromatic multicyclic systemmay be less favorable as this may increase the hydrophobicity and/or theaggregation tendency of the DNA-binder and therefore increase thehydrophobicity and/or the aggregation tendency of a compound of formula(I) or (II) and its conjugates. This may be especially true formulticyclic aromatic systems in which none or only one of the ring atomsis a heteroatom.

DNA binder DB2 may comprise an aromatic core structure. Alternatively,one or more rings may be non-aromatic and be either unsaturated orcompletely saturated.

A compound of formula (I) or (II) wherein ring B is connected to theDNA-alkylating unit via a vinyl group may contain a handle that allowsfor detoxification by means of for example oxidation or hydration of thedouble bond.

The moiety DB2 may for example be

wherein R^(8a), R^(9a), R^(10a), and R^(11a) have the same meaning asdefined for R⁸, R⁹, R¹⁰, and R¹¹, respectively, and are independentlyselected.

In a more specific embodiment, the moiety DB2 may for example be

wherein R⁷² and R⁷³ are independently selected from H and methyl.

In the exemplary structures of DB2, R⁸, R^(8a), R^(9a), R¹⁰, R^(10a),R¹¹, R^(11a), R¹⁵, R¹⁶, and R²¹ may for example each independently beselected to be H, be or contain another moiety selected from structuresDB1-DB9 or a derivative thereof, or be

wherein R⁶², R⁶³, R⁶⁴, and R⁶⁵ are independently selected from H, C₁₋₃alkyl, and

wherein jj, jj′, jj″, and jj′″ are independently selected from 0 to 8,X⁷⁴ is selected from

each tt, tt′, and tt″ is independently selected from 0 and 1, each X²¹and X²² is independently selected from O, S, NR⁶⁷, H₂, and C(R⁶⁷)R⁶⁸,wherein R⁶⁷ and R⁶⁸ are independently selected from H and optionallysubstituted C₁₋₃ alkyl or C₁₋₃ heteroalkyl, and R⁶⁶ is selected from H,COOH, CO₂Me, OH, OMe, NR⁶⁹R⁷⁰, NR⁶⁹C(O)CH₃, SH, SMe,

wherein X²³ is selected from halide, hydroxy, OC(O)R^(bb), andOC(O)OR^(bb), or C(O)—X²³ is an active ester, X²⁴ is selected fromhalide, mesyloxy, triflyloxy, and tosyloxy, R^(bb) is selected fromoptionally substituted C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, C₃₋₁₀ cycloalkyl,C₁₋₁₀ heterocycloalkyl, C₅₋₁₀ aryl, and C₁₋₁₀ heteroaryl, and R⁶⁹, R⁷⁰,and R^(7′) are independently selected from methyl and H.

In a further embodiment, the moiety DB2 may for example be

In another aspect of this invention, a compound of formula (I) or (II)has a DNA-binding unit of formula DB3 or DB4. These two moietiescomprise structures that are built up of an acetylene moiety coupled toa 5- or 6-membered ring. This ring may be aromatic or non-aromatic. Inthe latter case, it may be either unsaturated or completely saturated.Furthermore, the 5- or 6-membered ring may be fused to one or more otherrings to form an aromatic or non-aromatic ring system. Such a ringsystem is preferably flat as this may increase the DNA binding affinity.Either polar substituents or heteroatoms in the ring may provide forincreased water solubility and may favorably affect the pharmacologicalproperties of a compound of formula (I) or (II). The presence of anacetylene moiety in DNA-binding units DB3 and DB4 may provide for ahandle that allows detoxification by means of for example oxidation orhydration. The moiety DB3 may for example be

The moiety DB4 may for example be

In a more specific embodiment, the moiety DB3 may for example be

In another more specific embodiment, the moiety DB4 may for example be

wherein R⁷² is selected from H and methyl.

In the exemplary structures of DB3 and DB4, R⁸, R⁹, R¹⁰, R¹¹, and R²⁰may for example each independently be selected to be H, be or containanother moiety selected from structures DB1-DB9 or a derivative thereof,or be

wherein R⁶², R⁶³, R⁶⁴, and R⁶⁵ are independently selected from H, C₁₋₃alkyl, and

wherein jj, jj′, jj″, and jj′″ are independently selected from 0 to 8,X⁷⁴ is selected from

each tt, tt′, and tt″ is independently selected from 0 and 1, each X²¹and X²² is independently selected from O, S, NR⁶⁷, H₂, and C(R⁶⁷)R⁶⁸,wherein R⁶⁷ and R⁶⁸ are independently selected from H and optionallysubstituted C₁₋₃ alkyl or C₁₋₃ heteroalkyl, and R⁶⁶ is selected from H,COOH, CO₂Me, OH, OMe, NR⁶⁹R⁷⁰, NR⁶⁹C(O)CH₃, SH, SMe,

wherein X²³ is selected from halide, hydroxy, OC(O)R^(bb), andOC(O)OR^(bb), or C(O)—X²³ is an active ester, X²⁴ is selected fromhalide, mesyloxy, triflyloxy, and tosyloxy, R^(bb) is selected fromoptionally substituted C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, C₃₋₁₀ cycloalkyl,C₁₋₁₀ heterocycloalkyl, C₅₋₁₀ aryl, and C₁₋₁₀ heteroaryl, and R⁶⁹, R⁷⁰,and R⁷¹ are independently selected from methyl and H.

In a further embodiment, the moiety DB3 may for example be

In yet a further embodiment, the moiety DB4 may for example be

In another aspect of this invention, a compound of formula (I) or (II)has a DNA-binding unit of formula DB5. This moiety comprises structuresthat are built up of a 5-membered or 6-membered ring coupled to anoptionally substituted vinyl moiety. The 5-membered or 6-membered ringmay be aromatic or non-aromatic. In the latter case it may be eitherunsaturated or completely saturated. Polar substituents or heteroatomsin the ring and/or polar substituents on the vinyl group may provide forincreased water solubility and favorably affect the pharmacologicalproperties of a compound of formula (I) or (II). Aromatic substituentson the ring or vinyl moiety may increase the binding affinity. Thepresence of a vinyl moiety in DNA-binding unit DB5 may provide for ahandle that allows detoxification by means of for example oxidation orhydration.

The moiety DB5 may for example be

In the exemplary structures of DB5, R^(8b), R^(9b), and R¹⁵ may forexample each independently be selected to be H, be or contain anothermoiety selected from structures DB1-DB9 or a derivative thereof, or be

wherein R⁶², R⁶³, R⁶⁴, and R⁶⁵ are independently selected from H, C₁₋₃alkyl, and

wherein jj, jj′, jj″, and jj′″ are independently selected from 0 to 8,X⁷⁴ is selected from

each tt, tt′, and tt″ is independently selected from 0 and 1, each X²¹and X²² is independently selected from O, S, NR⁶⁷, H₂, and C(R⁶⁷)R⁶⁸,wherein R⁶⁷ and R⁶⁸ are independently selected from H and optionallysubstituted C₁₋₃ alkyl or C₁₋₃ heteroalkyl, and R⁶⁶ is selected from H,COOH, CO₂Me, OH, OMe, NR⁶⁹R⁷⁰, NR⁶⁹C(O)CH₃, SH, SMe,

wherein X²³ is selected from halide, hydroxy, OC(O)R^(bb), andOC(O)OR^(bb), or C(O)—X²³ is an active ester, X²⁴ is selected fromhalide, mesyloxy, triflyloxy, and tosyloxy, R^(bb) is selected fromoptionally substituted C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, C₃₋₁₀ cycloalkyl,C₁₋₁₀ heterocycloalkyl, C₅₋₁₀ aryl, and C₁₋₁₀ heteroaryl, and R⁶⁹, R⁷⁰,and R⁷¹ are independently selected from methyl and H.

In a further embodiment, the moiety DB5 may for example be

In another aspect of this invention, a compound of formula (I) or (II)has a DNA-binding unit of formula DB6 or DB7. These two moietiescomprise structures that are built up of two 5- or 6-membered rings thatare connected together via a direct single bond. These rings may eachindependently be aromatic or non-aromatic. In the latter case, they maybe either unsaturated or completely saturated. Furthermore, ring B maybe fused to one or more other rings to form an aromatic or non-aromaticring system, which is preferably flat. This may increase the DNA bindingaffinity. Either polar substituents or heteroatoms in one or more of therings may provide for increased water solubility and may favorablyaffect the pharmacological properties of a compound of formula (I) or(II).

The moiety DB6 may for example be

The moiety DB7 may for example be

wherein R^(8a), R^(9a), R^(10a), and R^(11a) have the same meaning asdefined for R⁸, R⁹, R¹⁰, and R¹¹, respectively, and are independentlyselected.

In a more specific embodiment, moiety DB6 may for example be

In another more specific embodiment, moiety DB7 may for example be

In the exemplary structures of DB6 and DB7, R⁸, R^(8a), R⁹, R^(9a), R¹⁰,R^(10a), R¹¹, R^(11a), R¹⁵, and R²⁰ may for example each independentlybe selected to be H, be or contain another moiety selected fromstructures DB1-DB9 or a derivative thereof, or be

wherein R⁶², R⁶³, R⁶⁴, and R⁶⁵ are independently selected from H, C₁₋₃alkyl, and

wherein jj, jj′, jj″, and jj′″ are independently selected from 0 to 8,X⁷⁴ is selected from

each tt, tt′, and tt″ is independently selected from 0 and 1, each X²¹and X²² is independently selected from O, S, NR⁶⁷, H₂, and C(R⁶⁷)R⁶⁸,wherein R⁶⁷ and R⁶⁸ are independently selected from H and optionallysubstituted C₁₋₃ alkyl or C₁₋₃ heteroalkyl, and R⁶⁶ is selected from H,COOH, CO₂Me, OH, OMe, NR⁶⁹R⁷⁰, NR⁶⁹C(O)CH₃, SH, SMe,

wherein X²³ is selected from halide, hydroxy, OC(O)R^(bb), andOC(O)OR^(bb), or C(O)—X²³ is an active ester, X²⁴ is selected fromhalide, mesyloxy, triflyloxy, and tosyloxy, R^(bb) is selected fromoptionally substituted C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, C₃₋₁₀ cycloalkyl,C₁₋₁₀ heterocycloalkyl, C₅₋₁₀ aryl, and C₁₋₁₀ heteroaryl, and R⁶⁹, R⁷⁰,and R⁷¹ are independently selected from methyl and H.

In a further embodiment, moiety DB6 may for example be

In another further embodiment, moiety DB7 may for example be

In another aspect of this invention, a compound of formula (I) or (II)has a DNA-binding unit of formula DB8. This moiety comprises structuresthat are built up of a monocyclic or multicyclic ring system coupled tothe DNA-alkylating unit via a methylene unit. Preferably, the DB8 moietycomprises a bicyclic ring system. The ring system may be aromatic ornon-aromatic. In the latter case it may be either unsaturated orcompletely saturated. Either polar substituents or heteroatoms in one ormore of the rings may provide for increased water solubility and mayfavorably affect the pharmacological properties of a compound of formula(I) or (II).

The moiety DB8 may for example be

wherein R^(8a), R^(9a), R^(10a), and R^(11a) have the same meaning asdefined for R⁸, R⁹, R¹⁰, and R¹¹, respectively, and are independentlyselected.

In the exemplary structures of DB8, R^(8a), R^(9a), R^(10a), R^(11a),R¹⁵, R^(15′), and R¹⁶ may for example each independently be selected tobe H, be or contain another moiety selected from structures DB1-DB9 or aderivative thereof, or be

wherein R⁶², R⁶³, R⁶⁴, and R⁶⁵ are independently selected from H, C₁₋₃alkyl, and

wherein jj, jj′, jj″, and jj′″ are independently selected from 0 to 8,X⁷⁴ is selected from

each tt, tt′, and tt″ is independently selected from 0 and 1, each X²¹and X²² is independently selected from O, S, NR⁶⁷, H₂, and C(R⁶⁷)R⁶⁸,wherein R⁶⁷ and R⁶⁸ are independently selected from H and optionallysubstituted C₁₋₃ alkyl or C₁₋₃ heteroalkyl, and R⁶⁶ is selected from H,COOH, CO₂Me, OH, OMe, NR⁶⁹R⁷⁰, NR⁶⁹C(O)CH₃, SH, SMe,

wherein X²³ is selected from halide, hydroxy, OC(O)R^(bb), andOC(O)OR^(bb), or C(O)—X²³ is an active ester, X²⁴ is selected fromhalide, mesyloxy, triflyloxy, and tosyloxy, R^(bb) is selected fromoptionally substituted C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, C₃₋₁₀ cycloalkyl,C₁₋₁₀ heterocycloalkyl, C₅₋₁₀ aryl, and C₁₋₁₀ heteroaryl, and R⁶⁹, R⁷⁰,and R⁷¹ are independently selected from methyl and H.

In a further embodiment, moiety DB8 may for example be

In another aspect of this invention, a compound of formula (I) or (II)has a DNA-binding unit of formula DB9. This moiety comprises structuresthat are built up of a 5-membered ring that is directly connected to thenitrogen atom of the DNA-alkylating unit via a single bond. The5-membered ring may be connected or fused to one or more other rings toform a multicyclic ring system, which is preferably flat. This mayincrease the DNA binding affinity. The ring system may be aromatic ornon-aromatic. In the latter case it may be either unsaturated orcompletely saturated. Either polar substituents or heteroatoms in one ormore of the rings may provide for increased water solubility and mayfavorably affect the pharmacological properties of a compound of formula(I) or (II). In one embodiment, the DB9 moiety contains at least tworing heteroatoms.

The moiety DB9 may for example be

wherein R^(8a), R^(9a), R^(10a), and R^(11a) have the same meaning asdefined for R⁸, R⁹, R¹⁰, and R¹¹, respectively, and are independentlyselected.

In the exemplary structures of DB9, R^(8a), R^(9a), R^(10a), R^(11a),and R⁹ may for example each independently be selected to be H, be orcontain another moiety selected from structures DB1-DB9 or a derivativethereof, or be

wherein R⁶², R⁶³, R⁶⁴, and R⁶⁵ are independently selected from H, C₁₋₃alkyl, and

wherein jj, jj′, jj″, and jj′″ are independently selected from 0 to 8,X⁷⁴ is selected from

each tt, tt′, and tt″ is independently selected from 0 and 1, each X²¹and X²² is independently selected from O, S, NR⁶⁷, H₂, and C(R⁶⁷)R⁶⁸,wherein R⁶⁷ and R⁶⁸ are independently selected from H and optionallysubstituted C₁₋₃ alkyl or C₁₋₃ heteroalkyl, and R⁶⁶ is selected from H,COOH, CO₂Me, OH, OMe, NR⁶⁹R⁷⁰, NR⁶⁹C(O)CH₃, SH, SMe,

wherein X²³ is selected from halide, hydroxy, OC(O)R^(bb), andOC(O)OR^(bb), or C(O)—X²³ is an active ester, X²⁴ is selected fromhalide, mesyloxy, triflyloxy, and tosyloxy, R^(bb) is selected fromoptionally substituted C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, C₃₋₁₀ cycloalkyl,C₁₋₁₀ heterocycloalkyl, C₅₋₁₀ aryl, and C₁₋₁₀ heteroaryl, and R⁶⁹, R⁷⁹,and R⁷¹ are independently selected from methyl and H.

In a further embodiment, moiety DB9 may for example be

In one embodiment of this invention, the DB unit is DB1. In anotherembodiment, the DB unit is DB2. In yet another embodiment, the DB unitis DB3. In yet another embodiment, the DB unit is DB4. In yet anotherembodiment, the DB unit is DB5. In yet another embodiment, the DB unitis DB6. In yet another embodiment, the DB unit is DB7. In yet anotherembodiment, the DB unit is DB8. In yet another embodiment, the DB unitis DB9. In another embodiment, the DB unit is selected from DB1, DB2,DB3, DB4, DB5, DB6, and DB7. In another embodiment, the DB unit isselected from DB1, DB2, DB5, DB6, and DB7. In a further embodiment, DBis selected from DB1, DB2, DB6, and DB7. In yet a further embodiment, DBis selected from DB1 and DB2. In yet a further embodiment, DB isselected from DB6 and DB7.

In one embodiment, R⁵, R^(5′), R⁶, R^(6′), R⁷, and R^(7′) areindependently selected from H, OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN,C(O)NH₂, C(O)H, C(O)OH, halogen, R^(e), SR^(e), S(O)R^(e), S(O)₂R^(e),S(O)OR^(e), S(O)₂OR^(e), OS(O)R^(e), OS(O)₂R^(e), OS(O)OR^(e),OS(O)₂OR^(e), OR^(e), NHR^(e), N(R^(e))R^(f), ⁺N(R^(e))(R^(f))R^(g),P(O)(OR^(e))(OR^(f)), OP(O)(OR^(e))(OR^(f)), SiR^(e)R^(f)R^(g),C(O)R^(e), C(O)OR^(e), C(O)N(R^(e))R^(f), OC(O)R^(e), OC(O)OR^(e),OC(O)N(R^(e))R^(f), N(R^(e))C(O)R^(f), N(R^(e))C(O)OR^(f), andN(R^(e))C(O)N(R^(f))R^(g), wherein R^(e), R^(f), and R^(g) areindependently selected from H and optionally substituted(CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₁₅ alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅cycloalkyl, C₁₋₁₅ heterocycloalkyl, C₅₋₁₅ aryl, or C₁₋₁₅ heteroaryl,wherein ee is selected from 1 to 1000, X¹³ is selected from O, S, andNR^(f1), and R^(f1) and R^(e1) are independently selected from H andC₁₋₃ alkyl, two or more of R^(e), R^(f), and R^(g) optionally beingjoined by one or more bonds to form one or more optionally substitutedcarbocycles and/or heterocycles, or R⁵+R^(5′) and/or R⁶+R^(6′) and/orR⁷+R^(7′) are independently selected from ═O, ═S, ═NOR^(e3),═C(R^(e3))R^(e4), and ═NR^(e3), R^(e3) and R^(e4) being independentlyselected from H and optionally substituted C₁₋₃ alkyl, or R^(5′)+R^(6′)and/or R^(6′)+R^(7′) and/or R^(7′)+R^(14′) are absent, resulting in adouble bond between the atoms designated to bear R^(5′) and R^(6′),and/or R^(6′) and R^(7′), and/or R^(7′) and R^(14′), respectively, twoor more of R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′), R¹⁴, and R^(14′)optionally being joined by one or more bonds to form one or moreoptionally substituted carbocycles and/or heterocycles.

In another embodiment, R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹,R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′),R²¹, R^(21′), R²², and R²³ are each independently selected from H, OH,SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(h),SR^(h), S(O)R″, S(O)₂R^(h), S(O)OR″, S(O)₂OR^(h), OS(O)R^(h),OS(O)₂R^(h), OS(O)OR^(h), OS(O)₂OR^(h), OR^(h), NHR^(h), N(R^(h))R^(i),⁺N(R^(h))(R^(i))R^(j), P(O)(OR^(h))(OR^(i)), OP(O)(OR^(h))(OR^(i)),SiR^(h)R^(i)R^(j), C(O)R^(h), C(O)OR^(h), C(O)N(R^(h))R^(i), OC(O)R^(h),OC(O)OR^(h), OC(O)N(R^(h))R^(i), N(R^(h))C(O)R^(i), N(R^(h))C(O)OR^(i),and N(R^(h))C(O)N(R^(i))R^(j), wherein R^(h), R^(i), and R^(j) areindependently selected from H and optionally substituted(CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₁₅ alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅cycloalkyl, C₁₋₁₅ heterocycloalkyl, C₅₋₁₅ aryl, or C₁₋₁₅ heteroaryl, twoor more of R^(h), R^(i), and R^(j) optionally being joined by one ormore bonds to form one or more optionally substituted carbocycles and/orheterocycles, or R⁸+R^(8′) and/or R⁹+R^(9′) and/or R¹⁰+R^(10′) and/orR¹¹+R^(11′) and/or R¹⁵+R^(15′) and/or R^(15″)+R^(15′″) and/orR¹⁶+R^(16′) and/or R²⁰+R^(20′) and/or R²¹+R^(21′) are independentlyselected from ═O, ═S, ═NOR^(h1), ═C(R^(h1))R^(h2), and ═NR^(h1), R^(h1)and R^(h2) being independently selected from H and optionallysubstituted C₁₋₃ alkyl, two or more of R⁸, R^(8′), R⁹, R^(9′), R¹⁰,R^(10′), R¹¹, R^(11′), R¹⁵, R^(15′), R¹⁵″, R^(15′″), R¹⁶, R^(16′), R²⁰,R^(20′), R²¹, R^(21′), R²², and R²³ optionally being joined by one ormore bonds to form one or more optionally substituted carbocycles and/orheterocycles.

In another embodiment, X³ is not represented by —X^(3a) and X^(3b)—.

In a further embodiment, if DB is DB2 in a compound of formula (I) or(II), then X¹ is O.

In a further embodiment, if DB is DB2 in a compound of formula (I) or(II) and X³ is represented by —X^(3a) and X^(3b)—, then X¹ is O.

Any of the substituents present on any of the rings in DB1, DB2, DB3,DB4, DB5, DB6, DB7, DB8, and DB9 may be or comprise another DB1, DB2,DB3, DB4, DB5, DB6, DB7, DB8, or DB9 moiety or any other DNA-bindingmoiety. Such another DB moiety or DNA-binding moiety may be connected tothe first DB moiety via for example an amide or ketone linkage.

In one embodiment, at least one ring in the DNA-binding moiety isaromatic. In another embodiment, at least one ring system is aromatic.In yet another embodiment, all rings in the DNA-binding moiety arearomatic or form an aromatic ring system. In yet another embodiment, theDNA-binding moiety contains at least a bicyclic aromatic moiety.

Substituents R¹ to R²³ may assist in improving the pharmacologicalproperties of a compound of formula (I) or (II) or its conjugate, forexample, its water solubility. This may for example be achieved byselecting one or more of the substituents R¹, R⁵, R^(5′), R⁶, R^(6′),R⁷, R^(7′), R¹⁴, R^(14′), R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹,R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′),R²¹, R^(21′), R²², and R²³ to comprise or be an oligoethylene glycol orpolyethylene glycol moiety or a triazole moiety. Alternatively orsimultaneously, one or more of the substituents may comprise or be awater-soluble group. The presence of a water-soluble group may not onlyresult in enhanced water solubility, but may also prevent a compound offormula (I) or (II) from crossing a biological barrier, especially whenit is an apolar barrier, such as a cell membrane. This may beadvantageous, especially when a compound of formula (I) or (II) isdelivered into a targeted cell through conjugation to a targeting moietybefore it is released from the conjugate as the compound of formula (I)or (II) will be unable to leave the cell. Even active transport via forexample the P-glycoprotein pump may be (partially) impaired. When acompound of formula (I) or (II) is prematurely released from theconjugate, e.g., in the circulation, it may be unable or only moderatelyable to enter (non-targeted) cells aspecifically as its membranetranslocation capabilities may be impaired by the water-soluble group.This may lead to increased selectivity and therefore to fewer sideeffects. In addition, at least in some instances, for example when thewater-soluble group is positively charged under physiologicalconditions, the water-soluble group may also improve the bindingaffinity for DNA by means of favorable electrostatic interactions withthe negatively charged phosphate groups. A water-soluble group is agroup that imparts increased solubility on a compound of formula (I) or(II) and/or a conjugate thereof. In one embodiment, water solubility ofa compound of this invention carrying a water-soluble group is increasedby more than 100% compared to the compound lacking said water-solublegroup. In other embodiments, water solubility of a compound of thisinvention carrying a water-soluble group is increased by more than 75%or 50% or 25% or 10% compared to the compound lacking said water-solublegroup. The water-soluble group may also contribute to prevent or reduceaggregation of compounds of this invention or to reduce side effects.Examples of water-soluble groups include, but are not limited to, —NH₂,—NH—, —NHR^(s), —NR^(s)—, —N(R^(s))(R^(t)), —⁺N(R^(s))(R^(t))—,—⁺N(R^(s))(R^(t))(R^(u)), —COOH, —OP(O)(OH)₂, —OP(O)(OH)O—,—OP(O)(OR^(s))O—, —OP(O)(OH)OR^(s), —OP(O)(OR^(s))OR^(t), —P(O)(OH)₂,—P(O)(OH)O—, —P(O)(OR^(s))OH, —P(O)(OR^(s))O—, —P(O)(OR^(s))(OR^(t)),—OS(O)₂OH, —OS(O)₂O—, —OS(O)₂OR^(s), —S(O)₂OH, —S(O)₂O—, —S(O)₂OR^(s),—OS(O)OH, —OS(O)O—, —OS(O)OR^(s), —S(O)OH, —S(O)O—, —OS(O)—,—S(O)OR^(s), —OS(O)_(2l —, —OS(O)) ₂R^(s), —S(O)₂—, —S(O)₂R^(s),—OS(O)R^(s), —S(O)—, —S(O)R^(s), —(OCH₂CH₂)_(v′)OH, —(OCH₂CH₂)_(v′)O—,—(OCH₂CH₂)_(v′)OR^(s), a sugar moiety, a mono-, di-, or oligosaccharidemoiety, and an oligopeptide moiety, or a protonated or deprotonated formthereof and further any combination thereof, wherein R^(s), R^(t), andR^(u) are independently selected from H and optionally substituted C₁₋₃alkyl, two or more of R^(s), R^(t), and R^(u) optionally being joined byone or more bonds to form one or more carbocycles and/or heterocycles,and v′ is an integer selected from 2 to 1000. The water-soluble groupmay be at any position within a substituent or may constitute the wholesubstituent. The water-soluble group may for example be located at anyinterior position, be part of the main chain, be part of a ringstructure, be a functional group pending to the main chain or a ring, orbe placed at the position at which the substituent is attached to theremainder of the agent.

In one embodiment, at least one of R¹, R⁵, R^(5′), R⁶, R^(6′), R⁷,R^(7′), R¹⁴, R^(14′), R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹,R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′),R²¹, R^(21′), R²², and R²³ contains a water-soluble group.

In another embodiment, at least one of R⁶, R⁷, R¹⁴, R⁸, R⁹, and R¹⁰contains a water-soluble group.

In yet other embodiments, R⁸ or R⁹ or R¹⁰ or R⁶ or R⁷ or R¹⁴ contains awater-soluble group.

In one embodiment, the water-soluble group is a carboxylic acid group.

In another embodiment, the water-soluble group is an amino group.

In further embodiments, the water-soluble group is a primary orsecondary or tertiary or quaternary amino (ammonium) group. In otherembodiments, the water-soluble group is a primary or secondary ortertiary or quaternary aliphatic amino (ammonium) group.

In other embodiments, the water-soluble group is a phosphonate group ora phosphate group or a sulfonate group or a sulfate group or a glycolgroup or an oligoethylene glycol group or an polyethylene glycol group.

A compound of formula (I) or (II) may not have a reactive moietyincorporated in its structure. On the other hand, as becomes clear fromthe above, a reactive moiety may be present in its structure that allowsfor reaction of a compound of formula (I) or (II) with another moiety.For example, a compound of formula (I) or (II) may be reacted with atargeting moiety or a linker-targeting moiety construct, e.g., anantibody or an antibody fragment, or an antibody-linker construct or anantibody fragment-linker construct, to prepare a targeting moiety-agentconjugate in one or more steps, which may or may not be a conjugate offormula (III). Such a targeting moiety-agent conjugate may either becleavable or non-cleavable. The formation of a targeting moiety-agentconjugate may not only be carried out through chemical synthesis, butmay also occur in situ, i.e., upon administration of a compound offormula (I) or (II) in vivo. The compound of formula (I) or (II) may forexample bind to endogenous proteins, e.g., albumin, upon administration.

In one embodiment, a compound of formula (I) or (II) is

or an isomer thereof, or a mixture of isomers.

In a further embodiment, a compound of formula (I) or (II) is

or an isomer of one of these, or a mixture of isomers.

In a further embodiment, a compound of formula (I) or (II) is

or an isomer of one of these, or a mixture of isomers.

In yet a further embodiment, a compound of formula (I) or (II) is

or an isomer of one of these, or a mixture of isomers.

In yet a further embodiment, a compound of formula (I) or (II) is

or an isomer of one of these, or a mixture of isomers.

In yet a further embodiment, a compound of formula (I) or (II) is

or an isomer of one of these, or a mixture of isomers.

In yet a further embodiment, a compound of formula (I) or (II) is

or an isomer of one of these, or a mixture of isomers.

In yet a further embodiment, a compound of formula (I) or (II) is

or an isomer of one of these, or a mixture of isomers.

In yet a further embodiment, a compound of formula (I) or (II) is

or an isomer of one of these, or a mixture of isomers.

In yet a further embodiment, a compound of formula (I) or (II) is

or an isomer of one of these, or a mixture of isomers.

Conjugates, Linker-Agent Conjugates and Bifunctional Linkers

In another aspect, this invention relates to a conjugate of a compoundof formula (I) or (II) that can be converted in vivo in one or moresteps to a compound of formula (I) or (II), respectively. The conjugatemay also be converted to a derivative of a compound of formula (I) or(II) in which a part of the promoiety attached to a compound of formula(I) or (II) in the conjugate remains attached to the compound of formula(I) or (II) after in vivo conversion. An alternative way of looking atthis is that the remaining moiety of the linker is part of the compoundof formula (I) or (II).

These conjugates may favorably affect the pharmacological properties andother characteristics of a compound of formula (I) or (II). In oneembodiment, this invention relates to a conjugate comprising a compoundof formula (I) or (II) conjugated to at least one promoiety. In anotherembodiment, this invention relates to a conjugate comprising a compoundof formula (I) or (II) conjugated to a promoiety.

In a further embodiment, this invention relates to a compound of formula(III):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinV² is either absent or a functional moiety;each L² is independently absent or a linking group linking V² to L;each L is independently absent or a linking group linking L² to one ormore V¹ and/or Y;each V¹ is independently absent or a conditionally-cleavable orconditionally-transformable moiety, which can be cleaved or transformedby a chemical, photochemical, physical, biological, or enzymaticprocess;each Y is independently absent or a self-eliminating spacer system whichis comprised of 1 or more self-elimination spacers and is linked to V¹,optionally L, and one or more Z;each p and q are numbers representing a degree of branching and are eachindependently a positive integer;z is a positive integer equal to or smaller than the total number ofattachment sites for Z;each Z is independently a compound of formula (I), (II), (I′), or (II′)as defined hereinabove wherein one or more of X¹, R⁵, R^(5′), R⁶,R^(6′), R⁷, R^(7′), R¹⁴, R^(14′), R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′),R¹¹, R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰,R^(20′), R²¹, R^(21′), R²², and R²³ may optionally in addition besubstituted by or be a substituent of formula (V):

wherein each V^(2′), L^(2′), L′, V^(1′), Y′, Z′, p′, q′, and z′ has thesame meaning as defined for V², L², L, V¹, Y, Z, p, q, and z,respectively, and is independently selected, the one or moresubstituents of formula (V) being independently connected via Y′ to oneor more of X¹, R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′), R¹⁴, R^(14′), R⁸,R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵, R^(15′), R^(15″),R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′), R²¹, R^(21′), R²², R²³, and/or toone or more atoms bearing these R substituents;each Z is independently connected to Y through either X¹, an atom in R⁵,R^(5′), R⁶, R^(6′), R⁷, R^(7′), R¹⁴, R^(14′), R⁸, R^(8′), R⁹, R^(9′),R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″), R¹⁶,R^(16′), R²⁰, R^(20′), R²¹, R^(21′), R²², R²³, or an atom bearing any ofthese R substituents; andat least V² or a V¹ is present.

In a further aspect, this invention relates to a compound of formula(III), wherein

V² is present and selected to be a targeting moiety and there is atleast one group of formula (V) that contains a V^(1′) moiety and eithercomprises a V^(2′), L^(2′), or L moiety that contains aX¹⁴(CH₂CH₂O)_(gg)CH₂CH₂X¹⁴ moiety, wherein gg is selected from 3 to 1000and each X¹⁴ is independently selected from

or said same group of formula (V) comprises at least 2X¹⁴CH₂CH₂OCH₂CH₂X¹⁴ moieties, in which each X¹⁴ is independentlyselected.

In a further aspect, this invention relates to a compound of formula(III) that contains at least one X¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴ moiety,wherein ff is selected from 1 to 1000 and each X¹⁴ is independentlyselected from

It should be understood from formula (III) that L can be connected to V¹and/or to Y. If L is connected to Y, this means that both V¹ and L, aswell as one or more Z, are connected to Y. If L is connected to V¹, thismeans that V¹ and one or more Z are connected to Y. L may also beconnected to both V¹ and Y at the same time. If Y is absent, L isconnected to V¹ or, if V¹ is absent, L is directly connected to Z.

The V²(-L²-L(-(V¹—Y))_(p))_(q)(Z)_(z-1) and one or moreV^(2′)(-L^(2′)-L′(-(V^(1′)—Y′))_(p′))_(q′)(Z′)_(z′-1) moieties, whereinL(-(V¹—Y))_(p) indicates that L can be connected to V¹ and/or to Y,connected to Z are herein referred to as promoieties.

The present invention also relates to a compound of formula (IV):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinRM is a reactive moiety and L, V¹, Y, Z, p, and z are as definedhereinabove, except that L is now linking RM to one or more V¹ and/or Y,and V¹, Y, and Z may contain protecting groups, and the one or moreV^(2′)-L^(2′) moieties optionally present in Z as defined hereinabovemay optionally and independently be RM′ instead, which is a reactivemoiety, and wherein, if there is more than 1 reactive moiety in (IV),some or all reactive moieties are the same or different. Theselinker-agent conjugates of formula (IV) may or may not be consideredintermediates for compounds of formula (III). In a compound of formula(IV), RM must be present while V¹ may be either present or absent.

In a further aspect, the present invention relates to a compound offormula (IV), wherein RM is a reactive moiety selected from carbamoylhalide [—N(R)C(O)X], acyl halide [—C(O)X], active ester [—C(O)OR],anhydride [—C(O)OC(O)OR], α-haloacetyl [—C(O)CH₂X], α-haloacetamide[—N(R)C(O)CH₂X], maleimide, isocyanate [—N═C═O], isothiocyanate[—N═C═S], disulfide [—S—SR], thiol [—SH], hydrazine [—NH₂NH₂], hydrazide[—C(O)NH₂NH₂], sulfonyl chloride [—S(O)₂Cl], aldehyde [—C(O)H], methylketone [—C(O)CH₃], vinyl sulfone [—S(O)₂—CH═CH₂], halomethyl [—CH₂Cl],and methyl sulfonate [—CH₂OS(O)₂R], and wherein at least one group offormula (V), being part of Z, contains a V^(1′) moiety and eithercomprises a V^(2′), L^(2′), or L′ moiety that contains aX¹⁴(CH₂CH₂O)_(gg)CH₂CH₂X¹⁴ moiety, wherein gg is selected from 3 to 1000and each X¹⁴ is independently selected from

or said same group of formula (V) comprises at least 2X¹⁴CH₂CH₂OCH₂CH₂X¹⁴ moieties, in which each X¹⁴ is independentlyselected. These linker-agent conjugates of formula (IV) may or may notbe considered intermediates for compounds of formula (III). In such acompound of formula (IV), RM must be present.

In another embodiment, a compound of formula (IV) contains an RM moietyselected from carbamoyl halide [—N(R)C(O)X], acyl halide [—C(O)X],active ester [—C(O)OR], anhydride [—C(O)OC(O)OR], α-haloacetyl[—C(O)CH₂X], α-haloacetamide [—N(R)C(O)CH₂X], maleimide, isocyanate[—N═C═O], isothiocyanate [—N═C═S], disulfide [—S—SR], thiol [—SH],hydrazine [—NH₂NH₂], hydrazide [—C(O)NH₂NH₂], sulfonyl chloride[—S(O)₂Cl], aldehyde [—C(O)H], methyl ketone [—C(O)CH₃], vinyl sulfone[—S(O)₂—CH═CH₂], halomethyl [—CH₂C1], and methyl sulfonate[—CH₂OS(O)₂R].

The RM-L(-(V¹—Y))_(p)(Z)_(z-1) and one or moreRM′-L′(-(V^(1′)—Y′))_(p′)(Z′)_(z′-1) moieties, wherein L(-(V¹—Y))_(p)indicates that L can be connected to V¹ and/or to Y, connected to Z areherein referred to as promoieties.

In yet a further aspect, this invention relates to novel bifunctionallinkers that contain a cleavage site, a self-elimination spacer systemand two reactive moieties, one of which can be reacted with atherapeutic or diagnostic moiety, e.g. a compound of formula (II or(II), and the other of which can be reacted with a functional moiety,such as a targeting moiety. These bifunctional linkers can be used toprepare conjugates of formulae (III) and (IV) of this invention orsimilar compounds with different therapeutic or diagnostic moieties.

More specifically, this invention relates to a compound of formula(VIII):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinL, V¹, Y, RM, p, and z are as defined for a compound of formula (IV),and RM2 is a reactive moiety or a leaving group. RM and each RM2 areindependently selected. These bifunctional linkers of formula (VIII) mayor may not be considered intermediates for compounds of formula (III)and (IV). Furthermore, these compounds may be considered to beintermediates for conjugates and linker-agent conjugates similar tocompounds of formula (III) and (IV) in which the Z moiety is atherapeutic or diagnostic moiety different from a compound of formula(I), (II), (I′), or (II′), or a promoiety-containing derivative thereof.Therefore, in another aspect, this invention relates to a conjugate offormula (III) in which the one or more Z moieties are independently atherapeutic or diagnostic moiety. In yet another aspect, this inventionrelates to a linker-agent conjugate of formula (IV) in which the one ormore Z moieties are independently a therapeutic or diagnostic moiety.

It is noted that the separate X¹⁴ moieties in the —CH₂CH₂X¹⁴ moietiesthat may be present in a compound of formula (III), (IV), or (VIII) areindependently selected.

It is also noted that z does not represent a degree of polymerization;hence z does not indicate that a number of moieties Z or RM2 areconnected to one another.

It is further noted that if Y or Y′ is connected to an atom of Z or RM2bearing a specific R substituent instead of to this R substituentitself, this in fact means that this R substituent is absent if this isnecessary to meet valency rules.

It is further noted that if X¹⁴ in for example —CH₂CH₂X¹⁴ represents

, then —CH₂CH₂X¹⁴ should be read as —CH₂CHX¹⁴.

It is noted that if a compound of this invention contains anoligoethylene glycol or polyethylene glycol moiety the actual number ofethylene glycol units may vary from molecule to molecule and thevariable designating the number of units actually represents the averagenumber of units. Although the average number of ethylene glycol units isgenerally limited to 1000 in the definitions of compounds of thisinvention, compounds with a larger average number of ethylene glycolunits are also encompassed by this invention.

It should be understood that this invention relates to enantiomericallypure and/or diastereomerically pure compounds of formulae (III), (IV),and (VIII) as well as to enantiomeric and/or diastereomeric mixtures ofcompounds of formulae (III), (IV), and (VIII).

When a compound of formula (III) or (IV) contains attachment sites in Yfor Z that are not coupled to Z, for instance as a consequence of anincomplete coupling reaction with Z during synthesis, these attachmentsites are considered to be attached to H, OH, a reactive moiety (e.g.,RM2), or a leaving group (e.g., RM2) instead. If all of said attachmentsites are connected to Z, then z equals the number of said attachmentsites; otherwise, z is lower. Compounds of this invention may exist as amixture, wherein each component of the mixture has a different z value.For example, the compound may exist as a mixture of two separatecompounds, one compound wherein z is 4 and another compound wherein z is3. Furthermore, for a given z, the compound may exist as a mixture of(constitutional) isomers as Z may be connected to distinct (sets of)attachment sites.

For reasons of clarity, when referring to the connections of one firstmoiety to other moieties within formula (III), (IV), or (VIII), ingeneral only those said other moieties are mentioned that are directlyattached to said first moiety in formula (III), (IV), or (VIII). Itshould be understood that if one of said other moieties is not present,said first moiety is actually connected to the moiety first in line thatis present, unless explicitly stated otherwise. For example, if it isstated that “V¹ is cleaved from Y”, this phrase actually means “V¹ iscleaved from Y, or from Z if Y is absent” and should be read as “V¹ iscleaved from Z” when reference is made to a compound lacking Y.

In a compound of formula (III) or (IV), Z may be conjugated to apromoiety through its water-soluble group, e.g., an oligoethylene glycolor polyethylene glycol moiety. In this way, the water-soluble group maycontribute less to the water solubility of the compound of formula (III)or (IV), but may contribute again to the water solubility of Z uponremoval of said promoiety.

In this document, whenever V², L², L, V¹, Y, Z, RM, p, q, or z ismentioned, it should be understood that the same can apply for eachV^(2′), L^(2′), L′, V^(1′), Y′, Z′, RM′, p′, q′, or z′, respectively,unless the context dictates otherwise.

The V¹ Moiety

In a compound of formula (III), (IV), or (VIII), the V¹ moiety is agroup that is conditionally cleavable or transformable. In other words,it is designed to be transformed and/or cleaved from Y by a chemical,photochemical, physical, biological, or enzymatic process upon beingbrought in or under a certain condition. This condition may for examplebe bringing a compound of the invention in an aqueous environment, whichleads to hydrolysis of V¹, or bringing a compound of the invention in anenvironment that contains an enzyme that recognizes and cleaves V¹, orbringing a compound of the invention under reducing conditions, whichleads to reduction and/or removal of V¹, or bringing a compound of theinvention under oxidizing conditions, which leads to oxidation and/orremoval of V¹, or bringing a compound of the invention in contact withradiation, e.g., UV light, which leads to transformation and/orcleavage, or bringing a compound of the invention in contact with heat,which leads to transformation and/or cleavage, or bringing a compound ofthe invention under reduced pressure, which leads to transformation,e.g., a retrocycloaddition, and/or cleavage, or bringing a compound ofthe invention under elevated or high pressure, which leads totransformation and/or cleavage. This condition may be met afteradministering a compound of this invention to an animal, e.g., a mammal,for example a human: the condition may be met when the compoundlocalizes to for example a specific organ, tissue, cell, subcellulartarget, or bacterial, viral, or microbial target, for example by thepresence of internal factors (e.g., target-specific enzymes or hypoxia)or application of external factors (e.g., radiation, magnetic fields) orthe condition may already be met directly upon administration (e.g.,ubiquitous enzymes in the circulation).

Cleavage of V¹ means that the bond between V¹ and Y is broken.Transformation of V¹ means that V¹ is converted into a different moietyand this event may directly or indirectly lead to self-cleavage of V¹from Y. Alternatively, transformation of V¹ may lead to formation of aV¹—Y moiety which is a self-immolative linker. In this case, Y onlybecomes self-immolative after transformation of V¹. The transformed V¹moiety actually becomes (partially) part of Y. For example, oxidation ofV¹ being a hydrogen atom to a hydroxyl group may lead to formation of apara- or ortho-hydroxybenzyloxycarbonyl V¹—Y moiety thatself-eliminates. As another example, reduction of V¹ being a nitro groupmay lead to formation of a para- or ortho-aminobenzyloxycarbonyl V¹—Ymoiety that self-eliminates.

Alternatively again, V¹ may be absent. In this instance, the promoietyis intended to be non-removable from Z and the whole promoiety or a partthereof (in case of degradation of a compound of formula (III) or (IV)at one or more other sites in the molecule) will stay connected to theone or more moieties Z. One alternative way to look at this is that thepart of the promoiety that remains attached to the moiety Z is in fact apart of moiety Z.

A compound of this invention may contain more than one V¹ moiety perpromoiety (p and/or q>1). These V¹ moieties may or may not be the sameand may or may not require the same conditions for transformation and/orcleavage.

The rate at which V¹ is transformed and/or cleaved may be dependent onthe other moieties in a compound of formula (III). For example, use of ashorter L and/or Y moiety may bring the functional moiety and/or Zcloser to the cleavage site, which may lead to a reduced rate oftransformation and/or cleavage. (Bulky) substituents on the L and/or Ymoiety may lead to a reduced rate of transformation and/or cleavage ofV¹ as well, especially when they are situated close to the site oftransformation/cleavage. Other effects, such as hydrogen bonding,neighboring group, and electronic effects, may play a role as well.

In one aspect of this invention, a conjugate is used to target one ormore moieties Z to target cells. In this instance, a V¹ moiety may forexample contain a substrate molecule that is cleaved by an enzymepresent in the vicinity of the target cells or inside the target cells,for example tumor cells. V¹ can for example contain a substrate that iscleaved by an enzyme present at elevated levels in the vicinity of orinside the target cells as compared to other parts of the body, or by anenzyme that is present only in the vicinity of or inside the targetcells.

It is important to recognize that if target site specificity is achievedsolely based upon the selective transformation and/or cleavage of saidV¹ at the target site, the condition causing the cleavage shouldpreferably, at least to a certain degree, be target site-specific,whereas the presence of another target-specific moiety in the compoundof the invention, for instance in a V² moiety, weakens or takes awaythis requirement. For example, when V² causes selective internalizationinto a target cell, an enzyme also present in other cells may transformand/or cleave V¹. However, cleavage should preferably not occur at asite distant from the target site. Therefore, the conjugate should notbe exposed to enzymes or conditions that can cause cleavage of V¹ atsites other than the target site. In one embodiment, transformationand/or cleavage of V¹ occur intracellularly. In another embodiment,transformation and/or cleavage of V¹ occur extracellularly. In anotherembodiment, transformation and/or cleavage of V¹ occur by a ubiquitousintracellular enzyme. In another embodiment, transformation and/orcleavage of V¹ occur by a ubiquitous extracellular enzyme.

In one embodiment, V¹ contains a single amino acid, a di-, tri-, tetra-,or oligopeptide, or a peptidomimetic, which consists of an amino acid oramino acid sequence or mimetic thereof recognized and cleavable by aproteolytic enzyme, for example plasmin, a cathepsin, cathepsin B,prostate-specific antigen (PSA), urokinase-type plasminogen activator(u-PA), or a member of the family of matrix metalloproteinases, presentin the vicinity of or inside the target cells, for example tumor cells.In one embodiment, V¹ is a peptide. In another embodiment, V¹ is asingle amino acid. In another embodiment, V¹ is a dipeptide. In anotherembodiment, V¹ is a tripeptide. In another embodiment, V¹ is atetrapeptide. In yet another embodiment, V¹ is a peptidomimetic. Apeptidomimetic may be an amino acid mimic or peptide mimic. An aminoacid mimic may for example be a derivative of a natural amino acid inwhich the amino group has been replaced by a hydroxy group or a triazolegroup, in which the α-amino group of the amino acid is alkylated, or inwhich the side chain is connected to the α-amino group instead of to theα-carbon. A peptide mimic may be a peptide that contains one or more ofsuch amino acid mimics.

In another embodiment, V¹ contains a β-glucuronide that is recognized byβ-glucuronidase present in the vicinity of or inside tumor cells.

In one embodiment, V¹ contains a substrate for an enzyme.

In another embodiment, V¹ is a substrate for an enzyme.

In one embodiment, V¹ contains a substrate for an extracellular enzyme.

In another embodiment, V¹ contains a substrate for an intracellularenzyme.

In yet another embodiment, V¹ contains a substrate for a lysosomalenzyme.

In yet another embodiment, V¹ contains a substrate for the serineprotease plasmin.

In yet another embodiment, V¹ contains a substrate for one or more ofthe cathepsins, for example cathepsin B.

In yet another embodiment, V¹ contains a substrate for a galactosidase.

In yet another embodiment, V¹ contains a substrate for quinone reductaseNQO1.

In yet another embodiment, V¹ contains a hydrazide, hydrazone or iminemoiety that is to be hydrolyzed intracellularly.

In yet another embodiment, V¹ contains a disulfide moiety that is to becleaved intracellularly.

When V¹ is cleaved extracellularly, the one or more Z moieties may bereleased extracellularly. This may provide the advantage that these Zmoieties are not only able to affect the cell(s) directly surroundingthe site of activation (e.g., target-positive cells), but also cellssomewhat further away from the site of activation (e.g., target-negativecells) due to diffusion (bystander effect), provided that the Z moietiesare able to penetrate the cell membrane.

An enzyme to cleave V¹ can also be transported to the vicinity of orinside target cells or target tissue via for example antibody-directedenzyme prodrug therapy (ADEPT), polymer-directed enzyme prodrug therapy(PDEPT), macromolecular-directed enzyme prodrug therapy (MDEPT),virus-directed enzyme prodrug therapy (VDEPT), or gene-directed enzymeprodrug therapy (GDEPT). In these approaches, the enzyme that needs tocleave V¹ is transported to or induced to be produced at the target sitebefore administration of the prodrug, e.g., a compound of formula (III)or (IV). In one embodiment, transformation and/or cleavage of V¹ occurthrough an enzyme linked to an antibody using the ADEPT approach.

In again another embodiment, V¹ contains a moiety, for example anitrobenzyl moiety that can be transformed and/or cleaved by reductionunder hypoxic conditions or by reduction by a nitroreductase. Afterreduction of the nitro group and cleavage of the resulting moiety viaself-elimination, self-elimination of the spacer system Y, if present,leads to release of one or more moieties Z.

In one embodiment, the invention relates to a conjugate wherein V¹ is asingle amino acid, a dipeptide, a tripeptide, a tetrapeptide, or anoligopeptide moiety comprised of natural L amino acids, unnatural Damino acids, or synthetic amino acids, or a peptidomimetic, or anycombination thereof.

In another embodiment, the invention relates to a compound wherein V¹comprises a tripeptide. The tripeptide may be linked via its C-terminusto Y. In one embodiment, the C-terminal amino acid residue of thetripeptide is selected from alanine, arginine, citrulline, and lysine,the middle amino acid residue of the tripeptide is selected fromalanine, valine, leucine, isoleucine, methionine, phenylalanine,cyclohexylglycine, tryptophan, and proline, and the N-terminal aminoacid residue of the tripeptide is selected from any natural or unnaturalamino acid.

In another embodiment, the invention relates to a compound wherein V¹comprises a dipeptide. The dipeptide may be linked via its C-terminus toY. In one embodiment, the C-terminal amino acid residue of the dipeptideis selected from alanine, arginine, citrulline, and lysine, and theN-terminal amino acid residue of the dipeptide is selected from anynatural or unnatural amino acid.

In yet another embodiment, the invention relates to a compound whereinV¹ comprises a single amino acid. The amino acid may be linked via itscarboxyl group to Y. In one embodiment, the amino acid is selected fromalanine, arginine, citrulline, and lysine.

In one embodiment, when the α-amino group of the N-terminal amino acidof V¹ is not coupled to L, this amino acid may be functionalized with asuitable blocking group coupled to the α-amino group or may be anunnatural amino acid such that undesired premature degradation of V¹ byfor example ubiquitous enzymes, e.g., exopeptidases, is prevented. Sucha blocking group may be any group that prevents or considerably retardspremature degradation of V¹. Examples of such a blocking group include aD-amino acid, an acetyl group, a tert-butyloxycarbonyl group, and anoligoethylene or polyethylene glycol.

In a further embodiment, V¹ is selected from D-alanylphenylalanyllysine,D-valylleucyllysine, D-alanylleucyllysine, D-valylphenylalanyllysine,D-valyltryptophanyllysine, D-alanyltrypto-phanyllysine,alanylphenylalanyllysine, valylleucyllysine, alanylleucyllysine,valylphenyl-alanyllysine, valyltryptophanyllysine,alanyltryptophanyllysine, D-alanylphenylalanylcitrulline,D-valylleucylcitrulline, D-alanylleucylcitrulline,D-valylphenylalanylcitrulline, D-valyl-tryptophanylcitrulline,D-alanyltryptophanylcitrulline, alanylphenylalanylcitrulline,valylleucyl-citrulline, alanylleucylcitrulline,valylphenylalanylcitrulline, valyltryptophanylcitrulline, andalanyltryptophanylcitrulline.

In yet another embodiment, V¹ is selected from phenylalanyllysine,valyllysine, valylalanine, D-phenylalanylphenylalanyllysine,phenylalanylphenylalanyllysine, glycylphenylalanyllysine, alanyllysine,valylcitrulline, N-methylvalylcitrulline, phenylalanylcitrulline, isoleucylcitrulline, tryptophanyllysine, tryptophanylcitrulline,phenylalanylarginine, phenylalanylalanine,glycylphenylalanylleucylglycine, alanylleucylalanylleucine,alanylarginylarginine, phenylalanyl-N⁹-tosylarginine,phenylalanyl-N⁹-nitroarginine, leucyllysine, leucylcitrulline, andphenylalanyl-O-benzoylthreonine.

In a further embodiment, V¹ is selected from phenylalanyllysine,valyllysine, and valylcitrulline.

In yet further embodiments, V¹ is phenylalanyllysine or valyllysine orvalylcitrulline.

Therefore, in one embodiment this invention relates to a compoundwherein V¹ contains a substrate that can be cleaved by a proteolyticenzyme, plasmin, a cathepsin, cathepsin B, β-glucuronidase, agalactosidase, prostate-specific antigen (PSA), urokinase-typeplasminogen activator (u-PA), a member of the family of matrixmetalloproteinases, or an enzyme localized by means of directed enzymeprodrug therapy, such as ADEPT, VDEPT, MDEPT, GDEPT, or PDEPT, orwherein V¹ contains a moiety that can be cleaved or transformed throughreduction under hypoxic conditions, through reduction by anitroreductase, or through oxidation.

In another aspect of this invention, a conjugate of this invention isused primarily to improve the pharmacological properties of Z. When apromoiety does not need to be selectively removed at a target site, V¹of said promoiety may for example be or contain a group that is cleavedby ubiquitous enzymes, e.g., esterases that are present in thecirculation or intracellular enzymes, such as for example proteases andphosphatases, by pH-controlled intramolecular cyclization, or byacid-catalyzed, base-catalyzed, or non-catalyzed hydrolysis, or V¹ mayfor example be or contain a disulfide or form a disulfide with aneighboring moiety. V¹ may therefore, optionally together with theconnecting atom(s) of L and/or Y, for example form a carbonate,carbamate, ureum, ester, amide, imine, hydrazone, hydrazide, oxime,disulfide, acetal, or ketal group that can be cleaved in vivo. Thismeans that V¹, optionally together with the connecting atom(s) of Land/or Y, can for example also represent —OC(O)—, —C(O)O—, —OC(O)O—,—N(R^(v))C(O)—, —C(O)N(R^(v))—, —N(R^(v))C(O)O—, —OC(O)N(R^(v))—,—N(R^(v))C(O)N(R^(w))—, —C(O)—, —OC(R^(v))(R^(w))—, —C(R^(v))(R^(w))O—,—OC(R^(v))(R^(w))O—, —C(R^(v))(R^(w))—, —S—, —S—S—, —C═, ═C—, —N═, ═N—,—C═N—, —N═C—, —O—N═, ═N—O—, —C═N—O—, —O—N═C—, —N(R^(v))—N═,═N—N(R^(v))—, —N(R^(v))—N═C—, or —C═N—N(R^(v))—, wherein R^(v) and R^(w)are independently selected from H and optionally substituted C₁₋₁₀alkyl, C₁₋₁₀ heteroalkyl, C₁₋₁₀ heteroaryl, C₃₋₁₀ cycloalkyl, C₁₋₁₀heterocycloalkyl, or C₅₋₁₀ aryl, R^(v) and R^(w) optionally being joinedby one or more bonds to form one or more optionally substitutedcarbocycles and/or heterocycles.

V¹ may therefore for example be or contain, optionally together with theconnecting atom(s) of L and/or Y, a peptide, an amino acid, apeptidomimetic, a disulfide, a monosaccharide or disaccharide or aderivative thereof, a nitroaromatic moiety, an imine, a hydrazide, or ahydrazone moiety. If V¹ or V¹—Y represents a whole promoiety or L isconnected to Y and not to V¹, V¹ may for example also be selected from amono-, di-, or oligosaccharide, R^(p)—[O(R^(p′)O)P(O)]_(pp)—,R^(p)—C(O)—, R^(p)—OC(O)—, and R^(p)—N(R^(p′))C(O)—, wherein pp isselected from 1 to 3 and each R^(p) and R^(p′) is independently selectedfrom H and optionally substituted C₁₋₁₅ alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅cycloalkyl, C₁₋₁₅ heterocycloalkyl, C₅₋₁₅ aryl, or C₁₋₁₅ heteroaryl,R^(p′) and R^(p′) optionally being joined by one or more bonds to formone or more optionally substituted carbocycles and/or heterocycles.

In one embodiment, V¹ is selected from phosphono, phenylaminocarbonyl,4-(piperidin-1-yl)piperidin-1-ylcarbonyl, piperazin-1-ylcarbonyl,piperidin-1-ylcarbonyl, pyrrolidin-1-ylcarbonyl, and4-methylpiperazin-1-ylcarbonyl.

V¹ itself may contribute to favorable pharmacological properties of theconjugate, for example through the presence of polar functional groupsin V¹.

If a conjugate of this invention contains more than 1 promoiety, one ofthese promoieties may be used to target the conjugate to a target site(targeting promoiety), whereas another promoiety is used to improve thepharmacological properties. In this instance, the V¹ moiety in thetargeting promoiety is preferably cleaved at the target site, forexample through a target site-specific process such as an enzymaticcleavage by an enzyme predominantly present at the target site orthrough a more generic intracellular process which can only occur aftertarget cell-selective internalization of the conjugate, whereas thepromoiety that helps to improve the pharmacological properties may becleaved either at the target site or systemically, for example byubiquitous enzymes.

It should be noted that V¹, either in the form of an amino acid, a di-,tri-, tetra-, or oligopeptide, or in any other form, may containprotecting groups. Compounds of the invention comprising such aprotected V¹ may not release any Z moiety when put under conditions thatwill transform and/or cleave the corresponding unprotected V¹. However,when said compounds are deprotected, such compounds will release one ormore Z moieties when put under the appropriate conditions. Compoundscomprising such a protected V¹ also fall under the scope of thisinvention. In one aspect the above can be envisioned for compounds offormula (IV). Suitable protecting groups for functional groups, inparticular for amino acids, are well-known to the organic chemist andmay for example be found in T. W. Greene, Protective Groups in OrganicSynthesis, John Wiley & Sons, New York, 1981. In another aspect, theprotecting group or protecting moiety may be present in a compound offormula (III) and this compound may be administered as such. This meansthat deprotection should occur in vivo before release of any Z can occurthrough transformation and/or cleavage of V¹. In vivo deprotection ofthe protected V¹ moiety in a compound of formula (III) may occur by, forexample, hydrolysis or enzymatic conversion. This deprotection may occurat the target site or aspecifically.

Compounds of formulae (III) and (IV) can be designed to eventuallyrelease a compound of formula (I) or (II), or a compound of formula (I′)or (II′), after transformation and/or cleavage of the one or more V¹ andV^(1′) moieties. Release of a compound of formula (I) or (II), acompound of formula (I′) or (II′), or a derivative thereof (for exampledue to only partial degradation of the promoiety) from a conjugate ofthis invention via another mechanism is however not excluded from thisinvention.

In another aspect of this invention, a compound of formula (III)represents an intermediate for the preparation of a compound of formula(I) or (II) or another compound of formula (III). In this instance, forexample, V², L², L, and Y are absent, p, q, and z all are 1, and the V¹moiety may be a protecting group. There may or may not be one or moreV^(2′)(-L^(2′)-L′(-(V^(1′)—Y′))_(p′))_(q′)(Z′)_(z′-1) moieties present,in which V^(2′), L^(2′), L′, and Y′ may or may not be absent, and p′,q′, and z′ all may or may not be 1. In one embodiment, a compound offormula (III) is a compound of formula (I) or (II) to which a V¹ moietyis attached. In another embodiment, a compound of formula (III) is acompound of formula (I) or (II) to which a V¹ moiety and aV^(2′)(-L^(2′)-L′(-(V^(1′)—Y′))_(p′)(Z′)_(z′-1) moiety are attached. Inyet another embodiment, a compound of formula (III) is a compound offormula (I) or (II) to which a V¹ moiety and a V^(1′) moiety areattached.

In one embodiment, V¹ is not a protecting group.

In another embodiment, V², L², L, and Y are absent, and p, q, and z allare 1.

In a further embodiment, V¹ is a chemically removable group.

In yet a further embodiment, V¹ is a chemically removable groupconnected to Z via X¹.

In yet another further embodiment, V¹ is a benzyl group connected to Zvia X¹.

In another embodiment, V¹ istert-butoxycarbonyl(methylamino)ethyl(methylamino)carbonyl.

In another embodiment, V¹ is4-(tert-butoxycarbonyl)piperazine-1-carbonyl.

In one embodiment, V¹ is connected to L via more than one functionalgroup on V¹.

In another embodiment, V¹ is connected to L via one functional group onV¹.

In another embodiment, V¹ is connected to L via a functional group inthe side chain of one of the natural or unnatural amino acids of V¹.

In another embodiment, the N-terminal amino acid of V¹ is connected viaits a amino group to L.

In another embodiment, V¹ is absent.

The Self-Eliminating Spacer System Y

The self-elimination spacer system Y, if present, links V¹ andoptionally L to one or more moieties Z, or RM2 in case of a compound offormula (VIII).

A self-elimination spacer system Y may be incorporated in a conjugate ofthis invention for example to improve the properties of Z or theconjugate in general, to provide for suitable coupling chemistries,and/or to create space between V¹ and Z.

A compound of this invention may contain more than one spacer system Yper promoiety. These moieties Y may or may not be the same.

After cleavage or transformation of V¹, the left-hand side of Y maybecome unblocked or a V¹—Y self-elimination moiety may be formed, whichresults in eventual release of one or more moieties Z. Theself-elimination spacer systems may for example be those described in WO02/083180 and WO 2004/043493, which are incorporated herein by referencein their entirety, the ones described in this section, as well as otherself-elimination spacers known to a person skilled in the art.

Moiety Y may, in addition to providing suitable coupling chemistries andcreation of space between the cleavage site and Z, help improve thepharmacological properties of the conjugate. For example, the presenceof a water-soluble moiety or substituent, such as a polyethylene glycolmoiety or a substituent that is charged, at least partly, atphysiological pH may contribute to the water solubility and/or increasethe storage stability and/or plasma stability of the conjugate.

In one aspect the invention is related to compounds wherein Y is

(W—)_(w)(X—)_(x)(A-)_(s),

whereinW and X are each a single-release 1,2+2n electronic cascade spacer(n≧1), being the same or different;A is an ω-amino amino carbonyl cyclization spacer that forms a cyclicureum derivative upon cyclization;s is 0 or 1;w and x are numbers representing degree of polymerization and areindependently an integer from 0 (included) to 5 (included).

In one embodiment, w+x is 0, 1, or 2. In another embodiment, s is 0. Inyet another embodiment, s is 1. In yet another embodiment, w+x is 1 or 2and s is 1. In yet another embodiment, w+x is 1 and s is 1. In yetanother embodiment, w is 1, x is 0, and s is 1.

According to a further embodiment of this invention, the 1,2+2nelectronic cascade spacers W and X are independently a moiety having theformula:

whereinQ′ is selected from —R¹¹⁰C═CR¹¹¹—, S, O, NR¹¹¹, —R¹¹¹C═N—, and—N═CR¹¹¹—;B is selected from NR¹¹², O, and S;

P is C(R¹⁰⁸)(R¹⁰⁹)Q;

R¹⁰⁶, R¹⁰⁷, B, and (T-)_(t)(T′-)_(t′)(T″-)_(t″P are connected to C)^(a), C^(b), C^(c), and C^(d) in such a way that B and(T-)_(t)(T′-)_(t′)(T″-)_(t″)P are connected to two adjacent carbon atomsor to C^(a) and C^(d), respectively;Q is absent or —O—C(O)—;t, t′, and t″ are numbers representing degree of polymerization and areindependently an integer from 0 (included) to 5 (included);T, T′, and T″ are independently selected from moieties having theformula:

R¹⁰⁶, R¹⁰⁷, R¹⁰⁸, R¹⁰⁹, R¹¹⁰, R¹¹¹, R¹¹², and R¹¹⁴ are independentlyselected from H, OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H,C(O)OH, halogen, R^(y), SR^(y), S(O)R^(y), S(O)₂R^(y), S(O)OR^(y),S(O)₂OR^(y), OS(O)R^(y), OS(O)₂R^(y), OS(O)OR^(y), OS(O)₂OR^(y), OR^(y),NHR^(y), N(R^(y))R^(y1), ⁺N(R^(y))(R^(y1))R^(y2), P(O)(OR^(y))(OR^(y1)),OP(O)(OR^(y))(OR^(y1)), C(O)R^(y), C(O)OR^(y), C(O)N(R^(y1))R^(y),OC(O)R^(y), OC(O)OR^(y), OC(O)N(R^(y))R^(y1), N(R^(y1))C(O)R^(y),N(R^(y1))C(O)OR^(y), and N(R^(y1))C(O)N(R^(y2))R^(y), wherein R^(y),R^(y1), and R^(y2) are independently selected from H and optionallysubstituted (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₂₀ alkyl, C₁₋₂₀heteroalkyl, C₃₋₂₀ cycloalkyl, C₁₋₂₀ heterocycloalkyl, C₅₋₂₀ aryl, orC₁₋₂₀ heteroaryl, wherein ee is selected from 1 to 1000, X¹³ is selectedfrom O, S, and NR^(f1), and R^(f1) and R^(e1) are independently selectedfrom H and C₁₋₃ alkyl, two or more of R^(y), R^(y1), and R^(y2)optionally being joined by one or more bonds to form one or moreoptionally substituted carbocycles and/or heterocycles, two or more ofthe substituents R¹⁰⁶, R¹⁰⁷, R¹⁰⁸, R¹⁰⁹, R¹¹⁰, R¹¹¹, R¹¹², R¹¹³, andR¹¹⁴ optionally being joined by one or more bonds to form one or moreoptionally substituted carbocycles and/or heterocycles.

In the formulae above, Q may be O—C(O), but it may also be absent. Forexample, a compound with a benzyl ether linkage between self-eliminationspacer and the group that leaves, the oxycarbonyl moiety thus beingabsent (Q is absent), has been reported to undergo self-elimination⁹.

In one embodiment, t, t′ and t″ are 0.

Substituents R¹⁰⁸ and R¹⁰⁹ can be used to tune the degree of shieldingof the linkage to the moiety attached to Q (e.g., A or Z). They may alsobe used to tune the rate at which the 1,2+2n electronic cascade spacersW and X self-eliminate. In one embodiment, both R¹⁰⁸ and R¹⁰⁹ are H. Inanother

Substituents R¹⁰⁶, R¹⁰⁷, R¹¹⁰, and R¹¹¹ can be used to tune the degreeof shielding of the linkage between V¹ and Y and may thus be used totune the cleavage rate of a compound of formula (III) or (IV).Furthermore, these substituents can be used to introduce additionalwater solubility into a compound of this invention. In some embodiments,the 1,2+2n electronic cascade spacer W or X is

wherein bb and cc are independently an integer from 0 to 10 and R¹²³ andR¹²⁴ are independently selected from H and methyl.

In another embodiment, the 1,2+2n electronic cascade spacer W or X is

According to a further embodiment of this invention, the ω-aminoaminocarbonyl cyclization elimination spacer A is a moiety having theformula:

whereinu is an integer of 0 or 1;R¹¹⁵, R¹¹⁶, R¹¹⁷, R¹¹⁸, R¹¹⁹, R¹²⁰, R¹²¹, and R¹²² are independentlyselected from H, OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H,C(O)OH, halogen, R^(z), SR^(z), S(O)R^(z), S(O)₂R^(z), S(O)OR^(z),S(O)₂OR^(z), OS(O)R^(z), OS(O)₂R^(z), OS(O)OR^(z), OS(O)₂OR^(z), OR^(z),NHR^(z), N(R^(z))R^(z1), ⁺N(R^(z))(R^(z1))R^(z2), P(O)(OR^(z))(OR^(z1)),OP(O)(OR^(z))(OR^(z1)), C(O)R^(z), C(O)OR^(z), C(O)N(R^(z1))R^(z),OC(O)R^(z), OC(O)OR^(z), OC(O)N(Rz)R^(z1), N(R^(z1))C(O)R^(z),N(R^(z1))C(O)OR^(z), and N(R^(z1))C(O)N(R^(z2))R^(z), wherein R^(z),R^(z1), and R^(z2) are independently selected from H and optionallysubstituted (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₂₀ alkyl, C₁₋₂₀heteroalkyl, C₃₋₂₀ cycloalkyl, C₁₋₂₀ heterocycloalkyl, C₅₋₂₀ aryl, orC₁₋₂₀ heteroaryl, wherein ee is selected from 1 to 1000, X¹³ is selectedfrom O, S, and NR^(f1), and R^(f1) and R^(e1) are independently selectedfrom H and C₁₋₃ alkyl, two or more of R^(z), R^(z1), and R^(z2)optionally being joined by one or more bonds to form one or moreoptionally substituted carbocycles and/or heterocycles, two or more ofthe substituents R¹¹⁵, R¹¹⁶, R¹¹⁷, R¹¹⁸, R¹¹⁹, R¹²⁰, R¹²¹, and R¹²²optionally being joined by one or more bonds to form one or moreoptionally substituted carbocycles and/or heterocycles.

R¹¹⁵ and R¹¹⁶ are substituent groups that are located on the nitrogenatoms that are part of the connecting linkages to the moieties adjacentto the co-amino aminocarbonyl cyclization elimination spacer in acompound of formula (III), (IV), or (VIII). The nitrogen bearing R¹¹⁶ isconnected to Z or RM2 via a carbonyl moiety. The nitrogen atom bearingR¹¹⁵ is connected to W, X, V¹, or L. The size and nature of substituentsR¹¹⁵ and R¹¹⁶ may, in addition to affecting pharmacological propertiesof a compound of formula (III), (IV), or (VIII) in general, especiallyaffect the stability of said connecting linkages. For example, choosingR¹¹⁶ to be a small substituent may in general cause the linkage to Z tobe less stable to hydrolysis or enzymatic degradation than when R¹¹⁶ isa more bulky substituent (e.g., a substituent that is branched at thecarbon α or β to the nitrogen atom). The nature of the R¹¹⁶ substituent,e.g., whether polar or apolar or whether or not charged at physiologicalpH, may affect the stability of the linkage to Z as well, because thesubstituent may affect the substrate properties of the connectinglinkage for specific enzymes or retard or increase the rate ofhydrolysis. If the connecting linkage is susceptible to enzymes that areubiquitously present, for example enzymes present in the circulation,this may cause premature degradation of the conjugate. The same holdsfor substituent R¹¹⁵ with regard to the connecting linkage to W, X, V¹,or L. Similarly, the substituents on the carbons directly attached tothe nitrogen atoms (R¹¹⁷, R¹¹⁸, R¹¹⁹, R¹²¹, and R¹²²) may contribute tothe stability of the connecting linkages.

Substituents R¹¹⁵ and R¹¹⁶ may also affect the cyclization rate of theco-amino aminocarbonyl cyclization elimination spacer A. Bulkysubstituents may hinder cyclization and thus generally reduce thecyclization rate. Small substituents may therefore in general befavorable for fast cyclization rates. However, other properties of thesubstituent, such as electronegativity, polarity, and availability ofhydrogen bond donor and/or acceptor groups, may also affect thecyclization rate. Furthermore, if only one of R¹¹⁵ and R¹¹⁶ is a bulkysubstituent, the cyclization rate may be enhanced with respect tocyclization spacers containing two non-bulky substituents, possiblybecause of B-strain. The cyclization rate may also be affected by theother substituents on the co-amino aminocarbonyl cyclization eliminationspacer. Properties of these substituents such as polarity,electronegativity, and hydrogen bonding capabilities, may have an effecton the cyclization rate. Furthermore, the presence of geminalsubstituents on one of the carbons in between the two nitrogen atoms mayenhance the cyclization rate as well (Thorpe-Ingold effect). The joinderof substituents to form one or more rings may also have an effect on thecyclization rate. In this respect, every modification that may bring thetwo nitrogen atoms closer together or that reduces entropy may have anaccelerating effect on the cyclization rate.

Effects on the cyclization rate may be pH dependent and the order of thecyclization rate of two ω-amino aminocarbonyl cyclization eliminationspacers may be reversed going from for example pH 7 to pH 5.

Although it may be beneficial in some cases for a conjugate of thisinvention to have an ω-amino aminocarbonyl cyclization eliminationspacer with a fast cyclization rate, thus releasing the active drugshortly after cleavage of V¹, in other cases it may be more beneficialto have an ω-amino aminocarbonyl cyclization elimination spacer that hasa slow cyclization rate or a cyclization rate that lies between twopredetermined limits or that preferably cyclizes at a fast rate onlywithin certain pH limits. This may cause the cyclization spacer-drugintermediate to survive for a certain amount of time as a prodrug thatwill release the drug slowly or after a certain condition has been met.

In one embodiment, R¹¹⁵ and R¹¹⁶ are independently selected from R^(z),wherein R^(z) is selected from H and optionally substituted(CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₂₀ alkyl, C₁₋₂₀ heteroalkyl, C₃₋₂₀cycloalkyl, C₁₋₂₀ heterocycloalkyl, C₅₋₂₀ aryl, and C₁₋₂₀ heteroaryl,wherein ee is selected from 1 to 1000, X¹³ is selected from O, S, andNR^(f1), and R^(f1) and R^(e1) are independently selected from H andC₁₋₃ alkyl.

In another embodiment, both R¹¹⁵ and R¹¹⁶ are not H. In anotherembodiment, both R¹¹⁵ and R¹¹⁶ are not methyl. In yet anotherembodiment, both R¹¹⁵ and R¹¹⁶ are not H and at least one of R¹¹⁵ andR¹¹⁶ is not methyl. In yet another embodiment, both R¹¹⁵ and R¹¹⁶ arenot H and R¹¹⁶ is not methyl. In yet another embodiment, at least one ofR¹¹⁵ and R¹¹⁶ is not H or methyl. Such cyclization spacers incorporatedin a compound of formula (III) or (IV) have been shown to have improvedproperties with respect to cyclization spacers in which R¹¹⁵ and R¹¹⁶are both selected from only H and methyl. For example, the rate ofself-elimination, the rate of drug release, conjugate stability, and/orconjugate polarity may be improved.

In one embodiment, R¹¹⁵ and R¹¹⁶ are independently selected fromoptionally substituted (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₆ alkyl, C₁₋₆heteroalkyl, C₃₋₆ cycloalkyl, C₁₋₆ heterocycloalkyl, C₅₋₆ aryl, and C₁₋₆heteroaryl, wherein ee is selected from 1 to 9, X¹³ is selected from O,S, and NR^(f1), and R^(f1) and R^(e1) are independently selected from Hand C₁₋₃ alkyl.

In another embodiment, R¹¹⁵ and R¹¹⁶ are independently selected from H,methyl, ethyl, isopropyl, tert-butyl, phenyl,(CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1),

wherein dd is selected from 0 to 10, dd′ is selected from 0 and 1, eachR¹²³ is independently selected from H and methyl, ee is selected from 1to 1000, X¹³ is selected from O, S, and NR^(f1), and R^(f1) and R^(e1)are independently selected from H and C₁₋₃ alkyl.

In further embodiments, R¹¹⁵ and R¹¹⁶ are independently selected from H,methyl, ethyl, isopropyl, tert-butyl, phenyl,(CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1),

wherein dd′ is selected from 0 and 1, each R¹²³ is independentlyselected from H and methyl, X¹³ is selected from O, S, and NR^(f1),R^(f1) and R^(e1) are independently selected from H and C₁₋₃ alkyl, ddis 1 or 2 or 3 or 4 or 5, and ee is 1 or 2 or 3 or 4 or 5 or 6 or 7 or8.

In another embodiment, R¹¹⁵ is methyl and R¹¹⁶ is selected from H,methyl, ethyl, isopropyl, tert-butyl, phenyl,(CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1),

wherein dd is selected from 0 to 10, dd′ is selected from 0 and 1, eachR¹²³ is independently selected from H and methyl, X¹³ is selected fromO, S, and NR^(f1), and R^(f1) and R^(e1) are independently selected fromH and C₁₋₃ alkyl, and ee is selected from 1 to 1000.

In another embodiment, R¹¹⁵ is methyl and R¹¹⁶ is selected from ethyl,isopropyl, tert-butyl, phenyl, (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1),

wherein dd is selected from 0 to 10, dd′ is selected from 0 and 1, eachR¹²³ is independently selected from H and methyl, X″ is selected from O,S, and NR^(f1), and R^(f1) and R^(e1) are independently selected from Hand C₁₋₃ alkyl, and ee is selected from 1 to 1000.

In yet another embodiment, R¹¹⁶ is methyl and R¹¹⁵ is selected from H,methyl, ethyl, isopropyl, tert-butyl, phenyl,(CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1),

wherein dd is selected from 0 to 10, dd′ is selected from 0 and 1, eachR¹²³ is independently selected from H and methyl, X¹³ is selected fromO, S, and NR^(f1), and R^(f1) and R^(e1) are independently selected fromH and C₁₋₃ alkyl, and ee is selected from 1 to 1000.

In yet a further embodiment, R¹¹⁵ and R¹¹⁶ are independently selectedfrom H, methyl, ethyl, isopropyl, tert-butyl, phenyl,

In yet a further embodiment, R¹¹⁵ and R¹¹⁶ are independently selectedfrom H, methyl, ethyl, isopropyl, tert-butyl, phenyl,

In yet a further embodiment, R¹¹⁵ is methyl and R¹¹⁶ is selected from H,methyl, ethyl, isopropyl, tert-butyl, phenyl,

In yet a further embodiment, R¹¹⁶ is methyl and R¹¹⁵ is selected from H,methyl, ethyl, isopropyl, tert-butyl, phenyl,

In yet another embodiment, R¹¹⁵ or R¹¹⁶ is joined with one of R¹¹⁷,R¹¹⁸, R¹¹⁹, R¹²⁰, R¹²¹, and R¹²² to form a saturated or unsaturatedring. In a further embodiment, R¹¹⁵ or R¹¹⁶ is joined with one of R¹¹⁷,R¹¹⁸, R¹¹⁹, R¹²⁰, R¹²¹, and R¹²² to form a saturated or unsaturated 5-or 6-membered ring. In yet a further embodiment, R¹¹⁵ is joined with oneof R¹¹⁷, R¹¹⁸, R¹¹⁹, R¹²⁰, R¹²¹, and R¹²² to form a saturated orunsaturated 5- or 6-membered ring. In yet a further embodiment, R¹¹⁶ isjoined with one of R¹¹⁷, R¹¹⁸, R¹¹⁹, R¹²⁰, R¹²¹, and R¹²² to form asaturated or unsaturated 5- or 6-membered ring.

In another embodiment, two of R¹¹⁷, R¹¹⁸, R¹¹⁹, R¹²⁰, R¹²¹, and R¹²² arejoined to form a saturated or unsaturated ring. In yet anotherembodiment, two of R¹¹⁷, R¹¹⁸, R¹¹⁹, R¹²⁰, R¹²¹, and R¹²² not beingconnected to the same carbon atom are joined to form a saturated orunsaturated 5- or 6-membered ring.

In one embodiment, the cyclization spacer A is selected from

In another embodiment, the cyclization spacer A is selected from

In a further embodiment, the cyclization spacer A is selected from

In a further embodiment, the cyclization spacer A is

In a further embodiment, the cyclization spacer A is selected from

wherein R¹¹⁵, R¹¹⁶, R¹¹⁷, R¹¹⁸, R¹¹⁹, R¹²⁰, R¹²¹, and R¹²² areindependently selected from H, OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN,C(O)NH₂, C(O)H, C(O)OH, halogen, R^(z), SR^(z), S(O)R^(z), S(O)₂R^(z),S(O)OR^(z), S(O)₂OR^(z), OS(O)R^(z), OS(O)₂R^(z), OS(O)OR^(z),OS(O)₂OR^(z), OR^(z), NHR^(z), N(R^(z))R^(z1), ⁺N(R^(z))(R^(z1))R^(z2),P(O)(OR^(z))(OR^(z1)), OP(O)(OR^(z))(OR^(z1)), C(O)R^(z), C(O)OR^(z),C(O)N(R^(z1))R^(z), OC(O)R^(z), OC(O)OR^(z), OC(O)N(R^(z))R^(z1),N(R^(z1))C(O)R^(z), N(R^(z1))C(O)OR^(z), andN(R^(z1))C(O)N(R^(z2))R^(z), wherein R^(z), R^(z1), and R^(z2) areindependently selected from H and optionally substituted(CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₂₀ alkyl, C₁₋₂₀ heteroalkyl, C₃₋₂₀cycloalkyl, C₁₋₂₀ heterocycloalkyl, C₅₋₂₀ aryl, or C₁₋₂₀ heteroaryl,wherein ee is selected from 1 to 1000, X¹³ is selected from O, S, andNR^(f1), and R^(f1) and R^(e1) are independently selected from H andC₁₋₃ alkyl, two or more of R^(z), R^(z1), and R^(z2) optionally beingjoined by one or more bonds to form one or more optionally substitutedcarbocycles and/or heterocycles, two or more of the substituents R¹¹⁵,R¹¹⁶, R¹¹⁷, R¹¹⁸, R¹¹⁹, R¹²⁰, R¹²¹, and R¹²² optionally being joined byone or more bonds to form one or more optionally substituted carbocyclesand/or heterocycles.

In yet a further embodiment, the cyclization spacer A is selected from

wherein R¹¹⁵ and R¹¹⁶ are independently selected from H, methyl, ethyl,isopropyl, tert-butyl, phenyl, (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1),

wherein dd is selected from 0 to 10, dd′ is selected from 0 and 1, eachR¹²³ is independently selected from H and methyl, ee is selected from 1to 1000, X¹³ is selected from O, S, and NR^(f1), and R^(f1) and R^(e1)are independently selected from H and C₁₋₃ alkyl,and wherein R¹¹⁷, R¹¹⁸, R¹¹⁹, R¹²⁰, R¹²¹, and R¹²² are independentlyselected from H, OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H,C(O)OH, halogen, R^(z), SR^(z), S(O)R^(z), S(O)₂R^(z), S(O)OR^(z),S(O)₂OR^(z), OS(O)R^(z), OS(O)₂R^(z), OS(O)OR^(z), OS(O)₂OR^(z), OR^(z),NHR^(z), ⁺N(R^(z))R^(z1), ⁺N(R^(z))(R^(z1))R^(z2),P(O)(OR^(z))(OR^(z1)), OP(O)(OR^(z))(OR^(z1)), C(O)R^(z), C(O)OR^(z),C(O)N(R^(z1))R^(z), OC(O)R^(z), OC(O)OR^(z), OC(O)N(R^(z))R^(z1),N(R^(z1))C(O)R^(z), N(R^(z1))C(O)OR^(z), andN(R^(z1))C(O)N(R^(z2))R^(z), wherein R^(z), R^(z1), and R^(z2) areindependently selected from H and optionally substituted(CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₂₀ alkyl, C₁₋₂₀ heteroalkyl, C₃₋₂₀cycloalkyl, C₁₋₂₀ heterocycloalkyl, C₅₋₂₀ aryl, or C₁₋₂₀ heteroaryl,wherein ee is selected from 1 to 1000, X¹³ is selected from O, S, andNR^(f1), and R^(f1) and R^(e1) are independently selected from H andC₁₋₃ alkyl.

In yet a further embodiment, the cyclization spacer A is selected from

wherein R¹¹⁵ and R¹¹⁶ are independently selected from H, methyl, ethyl,isopropyl, tert-butyl, phenyl,

and wherein R¹¹⁷, R¹¹⁸, R¹¹⁹, R¹²⁰, R¹²¹, and R¹²² are independentlyselected from H, OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H,C(O)OH, halogen, R^(z), SR^(z), S(O)R^(z), S(O)₂R^(z), S(O)OR^(z),S(O)₂OR^(z), OS(O)R^(z), OS(O)₂R^(z), OS(O)OR^(z), OS(O)₂OR^(z), OR^(z),NHR^(z), N(R^(z))R^(z1), ⁺N(R^(z))(R^(z1))R^(z2), P(O)(OR^(z))(OR^(z1)),OP(O)(OR^(z))(OR^(z1)), C(O)R^(z), C(O)OR^(z), C(O)N(R^(z1))R^(z),OC(O)R^(z), OC(O)OR^(z), OC(O)N(Rz)R^(z1), N(R^(z1))C(O)R^(z),N(R^(z1))C(O)OR^(z), and N(R^(z1))C(O)N(R^(z2))R^(z), wherein R^(z),R^(z1), and R^(z2) are independently selected from H and optionallysubstituted (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₂₀ alkyl, C₁₋₂₀heteroalkyl, C₃₋₂₀ cycloalkyl, C₁₋₂₀ heterocycloalkyl, C₅₋₂₀ aryl, orC₁₋₂₀ heteroaryl, wherein ee is selected from 1 to 1000, X¹³ is selectedfrom O, S, and NR^(f1), and R^(f1) and R^(e1) are independently selectedfrom H and C₁₋₃ alkyl.

In another embodiment, the cyclization spacer A is

wherein R¹¹⁵ and R¹¹⁶ are independently selected from H, methyl, ethyl,isopropyl, tert-butyl, phenyl, (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1),

wherein dd is selected from 0 to 10, dd′ is selected from 0 and 1, eachR¹²³ is independently selected from H and methyl, ee is selected from 1to 1000, X¹³ is selected from O, S, and NR^(f1), and R^(f1) and R^(e1)are independently selected from H and C₁₋₃ alkyl.

In another embodiment, the cyclization spacer A is

wherein R¹¹⁵ and R¹¹⁶ are independently selected from H, methyl, ethyl,isopropyl, tert-butyl, phenyl,

In another embodiment, the cyclization spacer A is

wherein R¹¹⁶ is selected from methyl, ethyl, isopropyl, tert-butyl,phenyl,

In another embodiment, the cyclization spacer A is

wherein R¹¹⁶ is selected from ethyl, isopropyl, tert-butyl, phenyl,

In another embodiment, the cyclization spacer A is selected from

In another embodiment, cyclization linker A is selected from

In other embodiments, the cyclization linker A is

In other embodiments, the cyclization linker A is

In one embodiment, Y is absent.

In another embodiment, this invention relates to a compound of formula(III) or (IV) wherein X¹ is O and Y is connected to X¹ via an w-aminoaminocarbonyl cyclization spacer being part of Y.

In one embodiment, the spacer system Y is selected from

In another embodiment, the spacer system Y is

In another embodiment, the spacer system Y is

In a further embodiment, the spacer system Y is

wherein A is selected from

wherein R¹¹⁵ and R¹¹⁶ are independently selected from H, methyl, ethyl,isopropyl, tert-butyl, phenyl, (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1),

wherein dd is selected from 0 to 10, dd′ is selected from 0 and 1, eachR¹²³ is independently selected from H and methyl, ee is selected from 1to 1000, X¹³ is selected from O, S, and NR^(f1), and R^(f1) and R^(e1)are independently selected from H and C₁₋₃ alkyl,and wherein R¹¹⁷, R¹¹⁸, R¹¹⁹, R¹²⁰, R¹²¹, and R¹²² are independentlyselected from H, OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H,C(O)OH, halogen, R^(z), SW, S(O)R^(z), S(O)₂R^(z), S(O)OR^(z),S(O)₂OR^(z), OS(O)R^(z), OS(O)₂R^(z), OS(O)OR^(z), OS(O)₂OR^(z), OR^(z),NHR^(z), N(R^(z))R^(z1), ⁺N(R^(z))(R^(z1))R^(z2), P(O)(OR^(z))(OR^(z1)),OP(O)(O)(OR^(z))(OR^(z1)), C(O)R^(z), C(O)R^(z), C(O)N(R^(z1))R^(z),OC(O)R^(z), OC(O)OR^(z), OC(O)N(R^(z))R^(z1), N(R^(z1))C(O)R^(z),N(R^(z1))C(O)OR^(z), and N(R^(z1))C(O)N(R^(z2))R^(z), wherein R^(z),R^(z1), and R^(z2) are independently selected from H and optionallysubstituted (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₂₀ alkyl, C₁₋₂₀heteroalkyl, C₃₋₂₀ cycloalkyl, C₁₋₂₀ heterocycloalkyl, C₅₋₂₀ aryl, orC₁₋₂₀ heteroaryl, wherein ee is selected from 1 to 1000, X¹³ is selectedfrom O, S, and NR^(f1), and R^(f1) and R^(e1) are independently selectedfrom H and C₁₋₃ alkyl.

In a further embodiment, the spacer system Y is

wherein A is selected from

wherein R¹¹⁶ is selected from methyl, ethyl, isopropyl, tert-butyl,phenyl,

In a further embodiment, the spacer system Y is

wherein A is selected from

wherein R¹¹⁶ is selected from ethyl, isopropyl, tert-butyl, phenyl,

In a further embodiment, the spacer system Y is

wherein A is selected from

In another embodiment, at least one of R¹¹⁵ and R¹¹⁶ is not H or methylwhen the two nitrogen atoms of the w-amino aminocarbonyl cyclizationspacer are connected via an unsubstituted ethylene bridge.

Other examples of self-eliminating spacers include, but are not limitedto, other spacers that can undergo cyclization¹⁰, such as optionallysubstituted 4-aminobutyric acid amides, appropriately substitutedbicyclo[2.2.1] and bicyclo[2.2.2] ring systems, 2-aminophenylpropionicacid amides, and “trimethyl-lock” cyclization spacers¹¹. A glycinespacer in which an amine-containing leaving group is connected at theα-position is another useful spacer for the compounds of theinvention.¹²

In a conjugate of this invention, a spacer system Y may be connected tomore than one V¹ moiety. In this case, transformation and/or cleavage ofone of these V¹ moieties may trigger the release of one or more Zmoieties. When V¹ moieties that are transformed or cleaved underdifferent conditions are connected to the same Y, release of one or moreZ moieties may occur when a conjugate of this invention is brought underone of several distinct conditions if Y can undergo self-elimination inmultiple ways. Alternatively, a spacer system Y may be used thatrequires to be triggered twice or even more times in order toself-eliminate. An example of such a self-elimination spacer is a bicinespacer.¹³ When such a spacer is used in combination with different,selectively cleavable V¹ moieties connected to said spacer, selectivityof release of Z may be increased as two different conditions must be metbefore Z is released.

The Linking Group L

The linking group L links one or more V¹ and/or Y moieties to L² or RM.Synthesis may be more straightforward when L is connected to V¹ insteadof Y and the compound may be less prone to premature degradation as V¹may be more shielded. Connection of L to Y may have the advantage thatV¹ may be transformed and/or cleaved with more ease. Other reasons toconnect L to Y may for example be that (part of) Y remains bound to Lupon cleavage of V¹, which prevents the release of reactive smallmolecules, and that the compound may display improved pharmacologicalproperties, solubility, or aggregation behavior. L may be absent, whichmeans that V¹ or Y is directly connected to either L² or RM. In anotheraspect, however, L is a linking group that functionally links or spacesthe one or more V¹ and/or Y moieties and the L² or RM moiety. In acompound of formula (IV), spacing may make the reactive moiety RM moreaccessible to the reaction partner, for example when the functionalmoiety V² is being coupled. In a compound of formula (III), spacing mayprovide for a better accessibility of V¹, because V² is further away,which, especially in the case of enzymatic cleavage or transformation ofV¹, may improve the rate at which V¹ is transformed and/or cleaved.However, it was found that a relatively short L moiety may improve invivo efficacy of a compound of formula (III) compared to similarcompounds with a relatively long L moiety, provided that V¹ can still betransformed and/or cleaved at the target site.

The linking group L must contain suitable functional groups at both ofits ends to provide for selective coupling with the one or more V¹and/or Y moieties and L² or RM.

The linking group L may be a water-soluble moiety or contain one or morewater-soluble moieties or water-soluble functional groups, such that Lcontributes to the water solubility of a compound of formula (III) or(IV). L may also be a moiety or contain one or more moieties thatreduce(s) aggregation of a compound of formula (III) or (IV), which mayor may not be a moiety/moieties that also increase(s) the watersolubility of a compound of formula (III) or (IV). Furthermore, L mayalso contain or be a moiety that causes a compound of formula (III) or(IV) to be less susceptible to an immune response or increased effluxfrom cells by multidrug resistance-associated transporter proteins. TheL moiety may contain an oligoethylene glycol or polyethylene glycolmoiety or a derivative thereof. This moiety may for example improve thewater solubility, reduce aggregation, reduce the immune response, and/orreduce the efflux from cells of a compound of formula (III) or (IV). Lmay for example contain a —(CH₂CH₂O)_(ff)CH₂CH₂X¹³— or—(CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1) moiety, wherein ff is selected from 1 to1000, X¹³ is selected from O, S, and NR^(f1), and R^(f1) and R^(e1) areindependently selected from H and C₁₋₃ alkyl. In one embodiment, such amoiety is part of a substituent attached to the main chain of L thatconnects RM or L² to V¹ and not present in the main chain of L itself inorder to keep the length of the main chain relatively short. The Lmoiety may also contain polar groups and/or groups that are charged, atleast partly, at physiological pH in order to improve thepharmacological properties of a compound of formula (III) or (IV).

In one aspect, the L moiety is a linear, branched, or dendritic moiety,so that it can be connected to one or more V¹ and/or Y moieties.Branching can occur via one or more cyclic structures or at one or morebranching atoms that may for example be carbon, nitrogen, silicon, orphosphorus. The number of branches in L that are connected to V¹ and/orY does not necessarily equal the total number of branches as in thecoupling reaction with V¹ and/or Y not all branches may be coupled to V¹and/or Y moieties due to incomplete chemical conversion. This means thatL may contain branches that are not coupled to V¹ or Y, but instead endin for example a functional group, H, OH, or a leaving group.

Therefore, when L is branched, compounds of this invention may exist asa mixture, wherein each component of the mixture has a different pvalue. For example, the compound may exist as a mixture of two separatecompounds, one compound wherein p is 2 and another compound wherein p is3. Furthermore, for a given p, the compound may exist as a mixture of(constitutional) isomers as V¹ and/or Y may be connected to distinct(sets of) branches on L.

In one embodiment, L is absent.

In another embodiment, L is a linear linker.

In another embodiment, the chain of atoms linking L² or RM to V¹consists of less than 19 atoms.

In another embodiment, the chain of atoms linking L² or RM to V¹consists of less than 15 atoms.

In another embodiment, the chain of atoms linking L² or RM to V¹consists of less than 10 atoms.

In another embodiment, the chain of atoms linking L² or RM to V¹consists of less than 5 atoms.

In another embodiment, L is a linear linker containing a 1,2,3-triazolemoiety. Such a linker may be built up through a cycloaddition reactionbetween a molecule containing an azide group and one containing anacetylene group.

In another embodiment, L is a linear linker containing a water-solublegroup. In another embodiment, L is a linear linker that comprises anoligoethylene glycol or polyethylene glycol or a derivative thereof. Ina further embodiment, L is a linear linker that contains a functionalgroup that is charged, at least partly, at physiological pH.

In another embodiment, L is a branched linker.

In another embodiment, L is a dendritic linker. The dendritic structuremay for example be built up through cycloaddition reactions betweenmolecules containing one or more azide groups and ones containing one ormore acetylene groups.

In one embodiment, p is 1.

In other embodiments, p is 2 or 3 or 4 or 6 or 8 or 9.

In another embodiment, L is represented by the formula:

whereinX¹⁰¹ and X¹⁰² are each independently O, NR¹³¹, or S;Each X¹⁰³ and X¹⁰⁴ is independently O, NR¹³², or S;Each xa, xb, xc, and xd is independently 0 or 1;kk is a number representing a degree of branching and is an integerselected from 1 (included) to 128 (included);ll is a number representing a degree of branching and is an integerselected from 0 (included) to 127 (included);

kk+ll≦128;

Each DD is independently H, OH, or a leaving group;R¹³⁰ is either a dendritic, branched, or unbranched multivalent moietyand selected from optionally substituted alkylene, oligoalkylene, orpolyalkylene, and optionally substituted heteroalkylene,oligoheteroalkylene, or polyheteroalkylene, and optionally substitutedarylene, oligoarylene, or polyarylene, and optionally substitutedheteroarylene, oligoheteroarylene, or polyheteroarylene, and optionallysubstituted cycloalkylene, oligocycloalkylene, or polycycloalkylene, andoptionally substituted heterocycloalkylene, oligoheterocycloalkylene, orpolyheterocycloalkylene, and —(CH₂CH₂O)_(v)—, -alkylene-(CH₂CH₂O)_(v)—,—(CH₂CH₂O)_(v)-alkylene-, -alkylene-(CH₂CH₂O)_(v)-alkylene-,-heteroalkylene-(CH₂CH₂O)_(v)—, —(CH₂CH₂O)_(v)-heteroalkylene-,-heteroalkylene-(CH₂CH₂O)_(v)-alkylene-,-heteroalkylene-(CH₂CH₂O)_(v)-heteroalkylene-,-alkylene-(CH₂CH₂O)_(v)-heteroalkylene-, X¹⁴(CH₂CH₂O)_(ff)CH₂CH₂X¹⁴, adendritic structure, a sugar residue, and an oligopeptide, or anycombination of two or more of the above, wherein optional substituentsmay for example be selected from C₁₋₈ alkyl, C₁₋₈ heteroalkyl, C₅₋₈aryl, C₁₋₈ heteroaryl, X¹⁴(CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1),—(CHOR^(133′))_(vv)—R¹³³, a sugar residue, and charged substituentsselected from SO₃ ⁻, OPO₃ ²⁻, PO₃ ²⁻, CO₂ ⁻, and NR¹³³R^(133′)R^(133″),or from any combination thereof;R¹³¹, R¹³², R¹³³, R^(133′), and R^(133″) are independently selected fromH, C₁₋₈ alkyl, and C₁₋₈ heteroalkyl;X₁₃ is selected from O, S, and NR^(f1);R^(f1) and R^(e1) are independently selected from H and C₁₋₃ alkyl;ee and ff are independently selected from 1 to 1000;

Each X¹⁴ is independently selected from

v is selected from 1 (included) to 1000 (included);vv is selected from 1 to 10.

In another embodiment, L is selected from

In yet another embodiment, L is selected from

In yet another embodiment, L is selected from

In yet another embodiment, L contains a moiety that may for example be

wherein ff is selected from 1 to 1000. In other embodiments, ff isselected from 3 to 1000 or 500 or 100 or 50 or 10. In other embodiments,ff is selected to be 1 or 2 or 3 or 4 or 5.

In yet another embodiment, L is selected from

wherein rr, rr′, rr″, and rr′″ each independently range from 0 to 8, X⁷⁴is selected from

each X⁴⁰ and X⁴¹ is independently selected from O, S, and NR¹³⁵, whereinR¹³⁵ is selected from H and C₁₋₃ alkyl, and each uu, uu′, and uu″ isindependently selected from 0 and 1.

In another embodiment, L is selected from

wherein X⁷⁰, X⁷¹, X⁷², and X⁷³ are independently selected from O, S, andNR⁸², X⁷⁴ is selected from

d is selected from 0 to 8, e is 0 or 1, gg′, gg″, gg′″, gg″″, and gg*are independently selected from 0 to 1000, and R⁸¹ and R⁸² areindependently selected from H and optionally substituted C₁₋₃ alkyl. Inother embodiments, gg′, gg″, gg′″, gg″″, and gg* are independentlyselected from 3 to 1000 or 500 or 100 or 50 or 10.

In yet another embodiment, L is not

The linkage between L and V¹ or Y may for example be an amide, a ureum,a carbonate, or a carbamate linkage. In one embodiment, the linkagebetween L and V¹ is a ureum, carbonate, or carbamate linkage.Alternatively, when V¹ is a peptide in which the N-terminal amino acidis an amino acid mimic that carries an α-azido group instead of anα-amino group, the linkage between L and V¹ may be a triazole groupformed through reaction of an acetylene group being part of L and theα-azido group of V^(i).

The Reactive Moiety RM and the Linking Group L²

The reactive moiety RM in a compound of formula (IV) is connected to thelinking group L and is able to react with a suitable functional group ona reaction partner.

In one embodiment of this invention, the reactive moiety RM is designedto react with a functional group on the moiety V², which results information of a compound of formula (III). In this reaction, the moietyRM is transformed into the moiety L². In another embodiment, thereactive moiety RM is designed to react with a complementary moiety insitu, e.g., in vivo, for example with serum albumin, to give a compoundthat may or may not be a compound of formula (III).

In one aspect of this invention, the reactive moiety RM contains anelectrophilic group that reacts with a nucleophilic group on thereaction partner, for example V², e.g., a thiol group, an amino group,or a hydroxy group.

In another aspect of this invention, the reactive moiety RM contains anucleophilic group that reacts with an electrophilic group on thereaction partner, for example V², e.g., an aldehyde group.

In another aspect of the invention, the reactive moiety RM contains acycloaddition partner moiety, e.g., an alkene, a diene, a 1,3-dipole, ora 1,3-dipolarophile, that reacts with a suitable complementarycycloaddition partner moiety on the reaction partner, for example V²,e.g., a diene, an alkene, a 1,3-dipolarophile, or a 1,3-dipole.

In another aspect of the invention, the reactive moiety RM contains agroup that can be coupled with a suitable complementary group on thereaction partner, for example V², under metal-catalyzed, biocatalyzed,or enzyme-catalyzed conditions, e.g., palladium-catalyzed conditions.

In one aspect of the invention, the reactive moiety RM is, withoutlimitation,

whereinX³⁵ is selected from halide, hydroxy, OC(O)R^(dd), and OC(O)OR^(dd), orC(O)—X³⁵ is an active ester, X³⁶ is selected from halide, mesyloxy,triflyloxy, and tosyloxy, and R^(dd) is selected from optionallysubstituted C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, C₃₋₁₀ cycloalkyl, C₁₋₁₀heterocycloalkyl, C₅₋₁₀ aryl, and C₁₋₁₀ heteroaryl.

In one embodiment, the moiety RM is selected from

which makes it able to react with a thiol group on the reaction partner,for example moiety V².

In another embodiment, the moiety RM is

which makes it able to react with a thiol group on the reaction partner,for example moiety V².

In another embodiment, the moiety RM is

wherein X³⁶ is Br, which makes it able to react with a thiol group onthe reaction partner, for example moiety V².

In another embodiment, the moiety RM is selected from

which makes it able to react with an amino group, e.g., a primary orsecondary amino group, on the reaction partner, for example moiety V².

In another embodiment, the moiety RM is selected from

which makes it able to react with an aldehyde group on the reactionpartner, for example moiety V².

The linking group L² in a compound of formula (III) represents theremainder of RM when the reactive moiety RM has reacted with V². Thisgroup then links the moiety V² with L. The group that remains may be abond, meaning that L² is absent. Typically, however, L² is a linkinggroup. When a compound of formula (III) is formed other than via acompound of formula (IV), L² does not represent the remainder of RM, butmay represent a similar or the same moiety and in addition be selectedfrom for example optionally substituted C₁₋₁₀ alkylene, C₁₋₁₀heteroalkylene, C₃₋₁₀ cycloalkylene, C₁₋₁₀ heterocycloalkylene, C₅₋₁₀arylene, and C₁₋₁₀ heteroarylene. The L² moiety may optionally comprisea X¹⁴(CH₂CH₂O)_(gg)CH₂CH₂X¹⁴ moiety.

In one embodiment, the moiety L² is absent.

In another embodiment, the moiety L² is, without limitation,

In a further embodiment, the moiety L² is

In yet a further embodiment, the moiety L² is

The RM2 Moiety

RM2 may be a reactive moiety or a leaving group.

If RM2 is a reactive moiety, it is preferably different from the RMreactive moiety. In this way, reactions with the bifunctional linker maybe carried out selectively and individually with each of the reactivemoieties. When RM2 is a reactive moiety, this means that after reactionwith a therapeutic or diagnostic moiety or a promoiety-containingderivative thereof, the RM2 moiety (or what remains of it afterreaction) may become part of said therapeutic or diagnostic moiety or apromoiety-containing derivative thereof optionally together with anypart of Y which may not self-eliminate because of a limited or absentleaving capability of therapeutic or diagnostic moiety-conjugated RM2.

In one embodiment, the RM2 may be connected to Y and be

whereinX³⁵ is selected from halide, hydroxy, OC(O)R^(dd), and OC(O)OR^(dd), orC(O)—X³⁵ is an active ester, X³⁶ is selected from halide, mesyloxy,triflyloxy, and tosyloxy, X⁹⁵ is either absent or selected from O, S,NR⁹⁵, C₁₋₃ alkyl, and C₁₋₃ heteroalkyl, R^(dd) is selected fromoptionally substituted C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, C₃₋₁₀ cycloalkyl,C₁₋₁₀ heterocycloalkyl, C₅₋₁₀ aryl, and C₁₋₁₀ heteroaryl, and R⁹⁵ isselected from H and C₁₋₃ alkyl.

RM2 may also be a leaving group. In this case, RM2 is replaced by thetherapeutic or diagnostic moiety or a promoiety-containing derivativethereof in a reaction of a compound of formula (VIII) with such atherapeutic or diagnostic moiety or a derivative thereof and thistherapeutic or diagnostic moiety or a derivative thereof becomesdirectly attached to Y.

In one embodiment, RM2 is selected from a halide (fluoride, chloride,bromide, and iodide), azide, a sulfonate (e.g., an optionallysubstituted C₁₋₆ alkanesulfonate, such as methanesulfonate,trifluoromethanesulfonate, and trifluoroethanesulfonate, or anoptionally substituted benzenesulfonate, such as p-toluenesulfonate andnosylate), imidazole, a cyclic imide thione, succinimide-N-oxide,phtalimide-N-oxide, p-nitrophenoxide, o-nitrophenoxide,pentafluorophenoxide, tetrafluorophenoxide, 1,3,5-trichlorophenoxide,1,3,5-trifluorophenoxide, a carboxylate, an aminocarboxylate(carbamate), an alkoxycarboxylate (carbonate), and an alkoxy group thattogether with the carbonyl group of Y can be referred to as an activeester group. Such alkoxy groups include, but are not limited to,succinimide-N-oxide, p-nitrophenoxide, pentafluorophenoxide,tetrafluorophenoxide, 1-hydroxybenzotriazole, and1-hydroxy-7-azabenzotriazole, and groups with comparable leavingcapability.

The Moiety V²

The moiety V² is a functional moiety, which means that it addsadditional functionality to a compound of the invention.

In one embodiment, V² is a targeting moiety. In another embodiment, theV² moiety is a moiety that improves the pharmacological properties of acompound of the invention. In yet another embodiment, the V² moiety is amoiety that causes accumulation of a compound of the invention at atarget site. In yet another embodiment, the V² moiety is a moiety thatimproves the aqueous solubility of a compound of the invention. In yetanother embodiment, the V² moiety is a moiety that increases thehydrophobicity of a compound of the invention. In yet anotherembodiment, the V² moiety is a moiety that reduces extravasation of acompound of the invention. In yet another embodiment, the V² moiety is amoiety that reduces excretion of a compound of the invention. In yetanother embodiment, the V² moiety is a moiety that reduces theimmunogenicity of a compound of the invention. In yet anotherembodiment, the V² moiety is a moiety that enhances the circulation timeof a compound of the invention. In yet another embodiment, the V² moietyis a moiety that enhances the ability of a compound of the invention tocross a biological barrier, e.g., a membrane, cell wall, or theblood-brain barrier. In yet another embodiment, the V² moiety is amoiety that enhances the ability of a compound of the invention tointernalize. In yet another embodiment, the V² moiety is a moiety thatenables a compound of the invention to internalize. In yet anotherembodiment, the V² moiety is a moiety that causes the compounds of theinvention to aggregate. In yet another embodiment, the V² moiety is amoiety that reduces aggregation of a compound of the invention. In yetanother embodiment, the V² moiety is a moiety that causes a compound ofthe invention to form micelles or liposomes. In yet another embodiment,the V² moiety is a moiety that causes complexation of a compound of theinvention to another molecule, e.g., a biomolecule. In yet anotherembodiment, the V² moiety is a polynucleotide moiety that complexes witha complementary nucleotide sequence, for example RNA or DNA. In yetanother embodiment, the V² moiety is a moiety that causes a compound ofthe invention to bind, associate, interact, or complex to anothermoiety, for example a (functionalized) surface or solid support.

In another embodiment, V² exhibits two or more different functions. TheV² moiety may for example be a targeting moiety and at the same timeimprove the pharmacological properties, including water solubility.

In one aspect of the invention, the moiety V² includes within its scopeany unit that binds or reactively associates or complexes with areceptor, a receptor complex, antigen, or other moiety associated with agiven target cell population. V² can be any molecule that binds to,complexes with, or reacts with a moiety of a cell population sought tobe therapeutically or otherwise biologically modified. The V² moietyacts to deliver the one or more moieties Z to the particular target cellpopulation with which V² reacts or to which V² binds. Such V² moietiesinclude, but are not limited to, aptamers, full-length antibodies andantibody fragments and derivatives thereof, lectins, biologic responsemodifiers, enzymes, vitamins, growth factors, steroids, nutrients, sugarresidues, oligosaccharide residues, hormones, and any derivativesthereof, or any combination of any of these. Upon binding, reactivelyassociating, or complexing, the compounds of the invention may or maynot be internalized. If internalization occurs, transformation and/orcleavage of V¹ preferably occur inside the target cell.

Useful non-immunoreactive protein, polypeptide, or peptide V² moietiesinclude, but are not limited to, transferrin, epidermal growth factors(“EGF”), bombesin, gastrin and its derivatives, gastrin-releasingpeptide, platelet-derived growth factor, IL-2, IL-6, transforming growthfactors (“TGF”), such as TGF-a and TGF-P, tumor growth factors, vacciniagrowth factor (“VGF”), insulin and insulin-like growth factors I and II,lectins, and apoprotein from low density lipoprotein.

Useful polyclonal antibody V² moieties are heterogeneous populations ofantibody molecules. Various procedures well-known in the art may be usedfor the production of polyclonal antibodies to an antigen-of-interest.

Useful monoclonal antibody V² moieties are homogeneous populations ofantibodies to a particular antigen (e.g., a cancer cell antigen). Amonoclonal antibody (mAb) to an antigen-of-interest can be prepared byusing any technique known in the art which provides for the productionof monoclonal antibody molecules.

Useful monoclonal antibody V² moieties include, but are not limited to,human monoclonal antibodies, humanized monoclonal antibodies, orchimeric human-mouse (or other species) monoclonal antibodies.Monoclonal antibodies may be made by any of numerous techniques known inthe art.

The V² moiety can also be a bispecific antibody. Methods for makingbispecific antibodies are known in the art.

The V² moiety can be a functionally active fragment, derivative, oranalog of an antibody that immunospecifically binds to an antigen on atarget cell, e.g., a cancer cell antigen. In this regard, “functionallyactive” means that the fragment, derivative, or analog is able to elicitanti-anti-idiotype antibodies that recognize the same antigen that theantibody from which the fragment, derivative, or analog is derived,recognizes.

Other useful V² moieties comprise fragments of antibodies including, butnot limited to, F(ab′)₂ fragments, which contain the variable region,the light chain constant region, and the CH1 domain of the heavy chain,which can be produced by pepsin digestion of the antibody molecule, andFab fragments, which can be generated by reducing the disulfide bridgesof the F(ab′)₂ fragments. Other useful V² moieties are heavy chain andlight chain dimers of antibodies, or any minimal fragment thereof suchas Fvs or single chain antibodies (SCAs), domain antibodies, anticalins,affibodies, nanobodies, and any other molecules with the same, similar,or comparable specificity as the parent antibody.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are usefulV² moieties. A chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine monoclonal and a humanimmunoglobulin constant region. Humanized antibodies are antibodymolecules from non-human species having one or more complementaritydetermining regions (CDRs) from the non-human species and a frameworkregion from a human immunoglobulin molecule.

Completely human antibodies are particularly desirable as V² moieties.Such antibodies can for example be produced using transgenic mice thatare incapable of expressing endogenous immunoglobulin heavy and lightchains genes, but which can express human heavy and light chain genes.

In other embodiments, the V² moiety is a fusion protein of an antibody,or a functionally active fragment or derivative thereof, for example onein which the antibody is fused via a covalent bond (e.g., a peptidebond) at either the N-terminus or the C-terminus to an amino acidsequence of another protein (or portion thereof, preferably at least a10, 20, or 50 amino acid portion of the protein) that is not theantibody. Preferably, the antibody or fragment thereof is covalentlylinked to the other protein at the N-terminus of the constant domain.

The V² moiety antibodies include analogs and derivatives that aremodified, i.e., by the covalent attachment of any type of molecule aslong as such covalent attachment permits the antibody to retain itsantigen-binding immunospecificity. For example, but not by way oflimitation, derivatives and analogs of antibodies include those thathave been further modified, e.g., by glycosylation, acetylation,pegylation, disulfide reduction, phosphylation, amidation,derivatization by known protecting or blocking groups, proteolyticcleavage, linkage to another protein, etc. Additionally, the analog orderivative can contain one or more unnatural amino acids.

The V² moiety antibodies include antibodies having modifications (e.g.,substitutions (for example cysteine to serine or serine to cysteine),deletions, or additions). In particular, they include antibodies havingmodifications in amino acid residues identified as involved in theinteraction between the Fc domain and the FcRn receptor. Modificationsmay also be introduced to be able to couple the antibody to linker-agentconjugates at specific positions on the antibody.

In a specific embodiment, an antibody immunospecific for a cancer ortumor antigen is used as a V² moiety in accordance with the compounds,compositions, and methods of the invention.

Antibodies immunospecific for a cancer cell antigen can be obtainedcommercially or produced by any method known to one of skill in the art,such as chemical synthesis or recombinant expression techniques. Thenucleotide sequences encoding antibodies immunospecific for a cancercell antigen can be obtained, e.g., from the GenBank database or adatabase like it, a commercial or other source, literature publications,or by routine cloning and sequencing.

Examples of antibodies available for the treatment of cancer that may beuseful for incorporation into conjugates of this invention as a V²moiety include, but are not limited to, HERCEPTIN (trastuzumab), whichis a humanized anti-HER2 monoclonal antibody for the treatment ofpatients with metastatic breast cancer; RITUXAN (rituximab), which is achimeric anti-CD20 monoclonal antibody for the treatment of patientswith non-Hodgkin's lymphoma; OvaRex (oregovomab), which is a murineantibody for the treatment of ovarian cancer; Panorex (edrecolomab),which is a murine IgG_(2a) antibody for the treatment of colorectalcancer; IMC-BEC2 (mitumomab), which is a murine IgG antibody for thetreatment of lung cancer; IMC-C225 (erbitux), which is a chimeric IgGantibody for the treatment of head and neck cancer; Vitaxin, which is ahumanized antibody for the treatment of sarcoma; Campath I/H(alemtuzumab), which is a humanized IgG₁ antibody for the treatment ofchronic lymphocytic leukemia (CLL); SGN-70, which is a humanizedanti-CD70 antibody for the treatment of hematologic malignancies; SmartMI95, which is a humanized IgG antibody for the treatment of acutemyeloid leukemia (AML); J591, which is a humanized antibody againstprostate specific membrane antigen; LymphoCide (epratuzumab), which is ahumanized IgG antibody for the treatment of non-Hodgkin's lymphoma;SGN-33, which is a humanized anti-CD33 antibody for the treatment ofacute myeloid leukemia; Smart ID 10, which is a humanized antibody forthe treatment of non-Hodgkin's lymphoma; Oncolym, which is a murineantibody for the treatment of non-Hodgkin's lymphoma; Allomune, which isa humanized anti-CD2 mAb for the treatment of Hodgkin's disease ornon-Hodgkin's lymphoma; Avastin (bevacizumab), which is a humanizedanti-VEGF antibody for the treatment of lung and colorectal cancers;SGN-40, which is a humanized anti-CD40 antibody for the treatment ofmultiple myeloma; SGN-30, which is a chimeric anti-CD30 antibody for thetreatment of Hodgkin's disease; CEAcide, which is a humanized anti-CEAantibody for the treatment of colorectal cancer; IMC-1C11, which is ananti-KDR chimeric antibody for the treatment of colorectal cancer, lungcancers, and melanoma; and Cetuximab, which is an anti-EGFR chimericantibody for the treatment of epidermal growth factor positive cancers.

Other antibodies that may be useful for incorporation into conjugates ofthis invention as a V² moiety include, but are not limited to,antibodies against the following antigens: CA125, CA9, CA6, CA15-3,CA19-9, L6, Lewis Y, Lewis X, Lewis A, alpha fetoprotein, CA 242,placental alkaline phosphatase, prostate specific antigen (PSA),prostate specific membrane antigen (PSMA), prostatic acid phosphatase,epidermal growth factor receptors, interleukin receptors, integrins,insulin-like growth factor receptors, CanAg, DAF, PEM, IRTA-2, IRTA-4,AFP, HER2, EGFR, VEGFR1, VEGFR2, MAGE-1, LUCA1, LUCA2, MAGE-2, MAGE-3,MAGE-4, ED-B, MADCAM, CEACAM5, MCP-1, Cripto, TAT226, VLA-4, C3B,anti-transferrin receptor, endosialin, E-selectin, GCC, GP-75,Syndecan-1, GPNMB, ROBO4, STEAP-1, CMET, EGP-1, Kim-1, Tim-1, Ephreceptor tyrosine kinases, HMW-MAA, TMEFF2, PSCA, CLL-1, TNF-α, FAP-α,IFN-α, EphA2, EphB2, EphB3, EphB4, EGFL-7, DLL-4, RS7, 4-1BB, TENB2,FLT3, p97, FGF19, FGFR2, glypican-3, P53, L53, RON, MN, GFR-α3, FDF03,TSLPR, MUC1-KLH, Tag 72, MUC18, B7H4, PTK7, RG-1, MUC16, CSAP, PDGF,PSMA, 5T4, EpCAM, SGA-1M, SGA-56M, SGA-72M, IGF1R, CCR2, CCR5, CTLA4,CLCA-1, ELAM1, DR5, CEA, CXCR-4, GD2, gp100, GD3 ganglioside, L243,HMGB1, GPC-3, MARTI, IL-2 receptor, CD2, CD3, CD303, CD4, CD20, CD43,CD44, CD30, CD55, CD151, CD154, CD19, CD21, CD23, CD79, CD52, CD25,CD45, CD46, CD56, CD59, CD7, CD137, CD138, CD74, CD133, CD80, CD63,CD64, CD66, CD140b, CD32, CD33, CD37, CD22, CD27, Apo-2, ERBB4, HLA-DR,HLA-DR10, human chorionic gonadotropin, CD38, CD40, CD70, mucin, P21, acancer stem cell-specific receptor, MPG, and Neu oncogene product. Manyother internalizing or non-internalizing antibodies that bind totumor-associated antigens can be used in this invention as a V² moiety,some of which have been reviewed¹⁴.

In one embodiment, the anti-Her2 antibody trastuzumab is selected as theV² moiety. In another embodiment, an epitope-binding functional fragmentor derivative of trastuzumab is selected as the V² moiety. In yetanother embodiment, an anti-Her2 antibody or a functional fragment orderivative thereof is selected as the V² moiety. In yet anotherembodiment, an anti-Her2 antibody or functional fragment or derivativethereof that has improved properties with respect to trastuzumab isselected as the V² moiety; improved properties may for example beincreased binding, longer circulation half-life, increasedinternalization rate, higher binding specificity for tumor tissuecompared to non-tumor tissue, and/or reduced immunogenicity.

In yet another embodiment, the anti-PSMA antibody J591 is selected asthe V² moiety. In another embodiment, an epitope-binding functionalfragment or derivative of J591 is selected as the V² moiety. In yetanother embodiment, an anti-PSMA antibody or a functional fragment orderivative thereof is selected as the V² moiety. In yet anotherembodiment, an anti-PSMA antibody or functional fragment or derivativethereof that has improved properties with respect to J591 is selected asthe V² moiety; improved properties may for example be increased binding,longer circulation half-life, increased internalization rate, higherbinding specificity for tumor tissue compared to non-tumor tissue,and/or reduced immunogenicity.

In yet other embodiments, an anti-CD19 antibody or an anti-CD22 antibodyor an anti-CD30 antibody or an anti-CD33 antibody or an anti-CD56antibody or an anti-CD70 antibody or an anti-CD74 antibody or ananti-CD138 antibody or an anti-CLL-1 antibody or an anti-5T4 antibody oran anti-CD303 antibody or an anti-Tag 72 antibody or an anti-Lewis Alike carbohydrate antibody or an anti-EphB3 antibody or an anti-HMW-MAAantibody or an anti-CD38 antibody or an anti-Cripto antibody or ananti-EphA2 antibody or an anti-GPNMB antibody or an anti-integrinantibody or an anti-MN antibody is selected as the V² moiety. In yetother embodiments, an epitope-binding functional fragment or derivativeof an anti-CD19 antibody or an anti-CD22 antibody or an anti-CD30antibody or an anti-CD33 antibody or an anti-CD56 antibody or ananti-CD70 antibody or an anti-CD74 antibody or an anti-CD138 antibody oran anti-CLL-1 antibody or an anti-5T4 antibody or an anti-CD303 antibodyor an anti-Tag 72 antibody or an anti-Lewis A like carbohydrate antibodyor an anti-EphB3 antibody or an anti-HMW-MAA antibody or an anti-CD38antibody or an anti-Cripto antibody or an anti-EphA2 antibody or ananti-GPNMB antibody or an anti-integrin antibody or an anti-MN antibodyis selected as the V² moiety. Therefore, in one embodiment, the V²moiety may be selected from an anti-CD19 antibody, an anti-CD22antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD56antibody, an anti-CD70 antibody, an anti-CD74 antibody, an anti-CD138antibody, an anti-CLL-1 antibody, an anti-5T4 antibody, an anti-CD303antibody, an anti-Tag 72 antibody, an anti-Lewis A like carbohydrateantibody, an anti-EphB3 antibody, an anti-HMW-MAA antibody, an anti-CD38antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-GPNMBantibody, an anti-integrin antibody, an anti-MN antibody, an anti-Her2antibody, and an anti-PSMA antibody, or from an epitope-bindingfunctional fragment or derivative of any of these.

In some embodiments, the antibody is an anti-nuclear antibody or anantibody that can bind to a receptor or receptor complex expressed on atarget cell. The receptor or receptor complex can comprise animmunoglobulin gene superfamily member, an integrin, a chemokinereceptor, a TNF receptor superfamily member, a cytokine receptor, amajor histocompatibility protein, a complement control protein, or alectin.

In another specific embodiment, an antibody immunospecific for anantigen associated with an autoimmune disease is used as a V² moiety inaccordance with the compounds, compositions, and methods of theinvention. In another specific embodiment, an antibody immunospecificfor a viral or microbial antigen is used as a V² moiety in accordancewith the compounds, compositions, and methods of the invention. As usedherein, the term “viral antigen” includes, but is not limited to, anyviral peptide, polypeptide, protein, saccharide, polysaccharide, orlipid that is capable of eliciting an immune response. As used herein,the term “microbial antigen” includes, but is not limited to, anymicrobial peptide, polypeptide, protein, saccharide, polysaccharide, orlipid that is capable of eliciting an immune response.

New antibodies are continually being discovered and developed, and thepresent invention provides that these new antibodies may also beincorporated into a compound of this invention.

V² can react with the reactive moiety RM via for example a heteroatom onV². Heteroatoms that may be present on V² include, without limitation,sulfur (in one embodiment, from a sulfhydryl group), oxygen (in oneembodiment, from a carboxyl or hydroxyl group), and nitrogen (in oneembodiment, from a primary or secondary amino group). V² may also reactvia for example a carbon atom (in one embodiment, from a carbonylgroup). These atoms can be present on V² in V²'s natural state, forexample a naturally occurring antibody, or can be introduced into V² via(chemical) modification.

Free sulfhydryl groups can be generated in an antibody or antibodyfragment by reduction of the antibody (fragment) with a reducing agentsuch as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). Inthis way, modified antibodies can be obtained that can have from 1 toabout 20 sulfhydryl groups, but typically between about 1 and about 9sulfhydryl groups.

Alternatively, V² can have one or more carbohydrate groups that can bechemically modified to contain one or more sulfhydryl groups. As anotheralternative, sulfhydryl groups can be generated by reaction of aminogroups, for example from lysine moieties, on V² with 2-iminothiolane(Traut's reagent), N-succinimidyl S-acetylthioacetate (SATA), or anothersulfhydryl-generating reagent. Such a reagent may also be used tointroduce additional functionality. For example, a sulfhydryl-generatingreagent may be used that not only introduces a sulfhydryl group onto V²,but at the same time introduces a water-soluble moiety such as anoligoethylene glycol or polyethylene glycol. Such a group may be presentas a substituent in the reagent (instead of being part of the mainchain) in order to keep the sulfhydryl group close to the V² moiety. Thepresence of such a water-soluble moiety may eventually improve thepharmacological properties of a compound of formula (III).

In one embodiment, the V² moiety is a receptor-binding moiety.

In another embodiment, the V² moiety is an antibody or an antibodyfragment or a derivative thereof.

In another embodiment, the V² moiety is a monoclonal antibody or afragment or derivative thereof.

In one embodiment, V² has one or more sulfhydryl groups and V² reactswith one or more RM moieties of one or more compounds of formula (IV)via one or more of these sulfhydryl groups' sulfur atoms to form acompound of formula (III) in which one or more compounds of formula (IV)have thus been incorporated.

In yet another embodiment, V² contains one or more disulfide bonds thatcan be chemically reduced to sulfhydryl groups (two for each disulfidebond), which can then be reacted with one or more reactive moieties RMto form a compound of formula (III).

In another embodiment, V² contains about 1 to about 3 sulfhydryl groups,which can be reacted with one or more reactive moieties RM to form acompound of formula (III).

In another embodiment, V² contains about 2 sulfhydryl groups, which canbe reacted with one or more reactive moieties RM to form a compound offormula (III).

In another embodiment, V² contains about 3 to about 5 sulfhydryl groups,which can be reacted with one or more reactive moieties RM to form acompound of formula (III).

In another embodiment, V² contains about 4 sulfhydryl groups, which canbe reacted with one or more reactive moieties RM to form a compound offormula (III).

In another embodiment, V² contains about 7 to about 9 sulfhydryl groups,which can be reacted with one or more reactive moieties RM to form acompound of formula (III).

In another embodiment, V² contains about 8 sulfhydryl groups, which canbe reacted with one or more reactive moieties RM to form a compound offormula (III).

In another embodiment, V² can have one or more carbohydrate groups thatcan be chemically modified to have one or more sulfhydryl groups. V²reacts with RM moieties via these one or more sulfhydryl groups' sulfuratoms to form a compound of formula (III).

In another embodiment, V² can have one or more lysine groups that can bechemically modified to have one or more sulfhydryl groups, which can bereacted with one or more reactive moieties RM to form a compound offormula (III).

Reactive moieties that can react with a sulfhydryl group include, butare not limited to, carbamoyl halide, acyl halide, α-haloacetamide,halomethyl ketone, vinyl sulfone, maleimide, and 2-disulfanylpyridine.

In yet another embodiment, V² can have one or more carbohydrate groupsthat can be oxidized to provide one or more aldehyde groups. Thecorresponding aldehyde(s) can then react with one or more reactivemoieties RM to form a compound of formula (III). Reactive moieties thatcan react with an aldehyde group on V² include, but are not limited to,hydrazine, hydrazide, amine, and hydroxylamine.

In yet another embodiment, V² can have one or more amino groups, e.g.,from lysine residues, which can be reacted with one or more reactivemoieties RM to form a compound of formula (III). Reactive moieties thatcan react with an amino group include, but are not limited to, carbamoylhalide, α-haloacetamide, acyl halide, aldehyde, sulfonyl chloride, alkylhalide, alkyl sulfonate, isocyanate, and isothiocyanate.

A conjugate of formula (III) may exist as a mixture, wherein eachcomponent of the mixture has a different q value. For example, thecompound may exist as a mixture of two separate compounds, one compoundwherein q is 2 and another compound wherein q is 3. As another example,a compound may exist as a mixture of 5 separate compounds, in which q is1, 2, 3, 4, and 5, respectively. As yet another example, a compound mayexist as a mixture of more than 5 separate compounds. Such mixturesmight further be “contaminated” with unconjugated V². When analyzing thecompound of formula (III) it is understood that q may be the (rounded)average number of L²-L(-(V¹—Y))_(p)(Z)_(z/g) units per V² moiety.Furthermore, for a given q, the compound may exist as a mixture of(constitutional) isomers as the q L²-L(-(V¹—Y))_(p)(Z)_(z/g) moietiesmay be connected to distinct (sets of) functional groups on V². Itshould be noted that the number of Z moieties in each unit only equalsz/q if all units are the same and/or contain the same number of Zmoieties.

In one embodiment, the V² moiety is connected to L² via a sulfur atom ofV².

In another embodiment, the V² moiety is connected to L² via a sulfuratom and q ranges from about 1 to about 20.

In another embodiment, the V² moiety is connected to L² via a sulfuratom and q ranges from about 1 to about 9.

In another embodiment, the V² moiety is connected to L² via a sulfuratom and q ranges from about 1 to about 3.

In another embodiment, the V² moiety is connected to L² via a sulfuratom and q is about 1.

In another embodiment, the V² moiety is connected to L² via a sulfuratom and q is about 2.

In another embodiment, the V² moiety is connected to L² via a sulfuratom and q ranges from about 3 to about 5.

In another embodiment, the V² moiety is connected to L² via a sulfuratom and q is about 4.

In another embodiment, the V² moiety is connected to L² via a sulfuratom and q ranges from about 7 to about 9.

In another embodiment, the V² moiety is connected to L² via a sulfuratom and q is about 8.

In one embodiment, a compound of formula (III) exists as a mixture ofseparate compounds.

In one embodiment, a compound of formula (III) exists as a mixture ofseparate compounds wherein q for three compounds is 1, 2, and 3,respectively.

In one embodiment, a compound of formula (III) exists as a mixture ofseparate compounds wherein q for three compounds is 3, 4, and 5,respectively.

In one embodiment, a compound of formula (III) exists as a mixture ofseparate compounds wherein q for three compounds is 5, 6, and 7,respectively.

In one embodiment, a compound of formula (III) exists as a mixture ofseparate compounds wherein q for three compounds is 7, 8, and 9,respectively.

In another embodiment, the V² moiety is connected to L² via a nitrogenatom of V².

In yet another embodiment, the V² moiety is connected to L² via a carbonatom of V².

In another aspect of this invention, the V² moiety includes any unitthat causes accumulation of compounds of the invention at the targetsite or in the vicinity thereof by a mechanism other than binding orreactively associating or complexing with a receptor, antigen, or otherreceptive moiety associated with a given target site, e.g., a targetcell population. One way to achieve this is for example to use a largemacromolecule as a V² moiety, which targets to solid tumor tissuethrough the enhanced permeability and retention (EPR) effect. Ringsdorfreported use of polymers to target antitumor agents to tumors.¹⁵ Throughthis EPR effect, macromolecules passively accumulate in solid tumors asa consequence of the disorganized pathology of angiogenic tumorvasculature with its discontinuous endothelium, leading tohyperpermeability to large macromolecules, and the lack of effectivetumor lymphatic drainage.

The V² moiety may for example be a branched or unbranched polymer, suchas for example poly[N-(2-hydroxypropyl)methacrylamide] (HPMA),hydroxyethyl starch (HES), poly(2-hydroxyethyl methacrylate) (HEMA),polyglutamic acid or poly-L-glutamic acid (PG), carboxymethyldextran(CMDex), a polyacetal, chitosan, a polypeptide, an oligoethylene glycolor polyethylene glycol (PEG), or a copolymer, such as an HPMA copolymer,an HPMA-methacrylic acid copolymer, a HEMA-methacrylic acid copolymer, aCMDex copolymer, a β-cyclodextrin copolymer, a PEG copolymer, or apoly(lactic-co-glycolic) acid copolymer.¹⁶ In this document both polymerand copolymer are referred to as polymer.

The polymer may be connected to L² via any suitable functional group,which can be located at one or both ends of the polymer, meaning that inthe conjugate q ranges from 1 to 2, or alternatively, the functionalgroups may (also) be located on groups pendant on the polymer such thatL² is (also) connected to the polymer via these pendant groups with qtypically ranging from 1 to about 1000. Optionally, the polymer may alsocontain an additional targeting group that can bind or reactivelyassociate or complex with a receptive moiety, e.g., an antibody orantibody derivative, bonded to the polymer either via a pendant group orend group, such that improved targeting to the target site is achieved.

Alternatively, the V² moiety may be a dendrimer or a protein or proteinfragment, e.g., serum albumin, which has no targeting properties exceptfor its ability to accumulate at the target site because of its size ormolecular weight.

In one embodiment, the V² moiety contains a polymer.

In another embodiment, the V² moiety is a polymer.

In another embodiment, the V² moiety is a polymer and q ranges from 1 toabout 1000.

In other embodiments, the V² moiety is a polymer and q ranges from 1 toabout 500 or 400 or 300 or 200 or 100 or less than 100.

In another embodiment, the V² moiety is a polymer and q ranges from 1 to2.

In another embodiment, the V² moiety is a polymer and q is 1.

In a specific embodiment, the V² moiety is an oligoethylene glycol or apolyethylene glycol or a derivative thereof.

In another embodiment, the V² moiety is a dendrimer, a protein, or aprotein fragment.

In another embodiment, V² is absent.

In another embodiment, the V² moiety is a moiety that is able totransport the conjugate across a biological barrier, e.g., a cellmembrane, either with or without prior binding, associating, orcomplexing with a receptor or receptor complex. In one embodiment, theV² moiety is a Tat peptide or a derivative, fragment, or analog thereof,or a moiety that has similar transmembrane delivery properties. Inanother embodiment, the V² moiety is a protein or protein fragment, anantibody or an antibody fragment, a receptor-binding or peptide vectormoiety, or a polymeric or dendritic moiety, or any combination thereof,to which is attached a Tat peptide or a derivative, fragment, or analogthereof, or a moiety that has similar transmembrane delivery properties.

Thus, in one aspect of the invention, the moiety V² is a targetingmoiety and is selected from the group consisting of a protein or proteinfragment, an antibody or an antibody fragment, a receptor-binding orpeptide vector moiety, and a polymeric or dendritic moiety, and anycombination or derivative thereof.

In another aspect of the invention, the V² moiety is a moiety thatimproves the pharmacological properties of a conjugate of the invention.For example, the moiety V² can be chosen such that the water solubilityof the conjugate is (further) improved. This can be achieved by choosingV² to be a hydrophilic moiety. Alternatively, the V² moiety can be usedfor example to increase the residence time of the compound in thecirculation, to reduce extravasation and/or excretion, to reduceaggregation, and/or to reduce the immunogenicity of the compound. Thismay for example be achieved by choosing V² to be or contain apolyethylene glycol or oligoethylene glycol or derivative thereof. Whenthe moiety V² is a moiety that improves the pharmacological propertiesof a compound of the invention and V¹ is a moiety that can be cleaved ortransformed aspecifically and there are no V^(1′) and V^(2′) moieties,the compound solely serves to improve the (pharmacological) propertiesof the one or more Z moieties.

In one embodiment, V² is a moiety that improves the pharmacologicalproperties and V¹ is a moiety that can be cleaved or transformedspecifically.

In another embodiment, V² is an oligoethylene glycol or a polyethyleneglycol or a derivative thereof and V¹ is a moiety that can be cleaved ortransformed specifically.

In another embodiment, V² is a moiety that improves the pharmacologicalproperties and V¹ is a moiety that can be cleaved or transformedaspecifically.

In another embodiment, V² is an oligoethylene glycol or a polyethyleneglycol or a derivative thereof and V¹ is a moiety that can be cleaved ortransformed aspecifically.

In another embodiment, V² is an oligoethylene glycol or a polyethyleneglycol or a derivative thereof and V¹ is a moiety that can be cleaved byubiquitous enzymes.

In another embodiment, V² is an oligoethylene glycol or a polyethyleneglycol or a derivative thereof and V¹ is a hydrolyzable moiety.

In another embodiment, V² contains a X¹⁴(CH₂CH₂O)_(gg)CH₂CH₂X¹⁴ moiety.

In one aspect of this invention, the V² moiety is represented by formula(VI):

wherein V²*, L²*, L*, V¹*, Y*, p*, q*, and z* have the same meaning asV², L², L, V¹, Y, p, q, and z, respectively, as defined in this documentand are selected independently, except that Y* is connected to L². Itshould be noted that z* actually equals q, assuming that all Y* areindeed connected to L². When a compound of formula (III) contains a V²moiety represented by formula (VI), the one or more L² moieties are thusconnected to Y.

Use of a V² moiety of formula (VI) in a conjugate of formula (III)implicates that two conditionally-cleavable orconditionally-transformable moieties may be present in between thefunctional moiety V²* and Z, and therefore two separatecleavages/transformations may be required to release Z. The requirementthat two different conditions need to have been met—in consecutiveorder—before one or more Z are released might favorably affect theproperties of the conjugate. For instance, it may increase the targetingefficiency and therapeutic index of the conjugate. The twotransformations/cleavages may occur at differentextracellular/intracellular locations. The moiety to be removed by thesecond cleavage or as a consequence of the second transformation may forexample be used to help transport Z from a first extracellular orintracellular location (where the first cleavage has occurred) to asecond extracellular or intracellular location, or to stabilize Z untilit is closer to its target, or to (temporarily) increase the watersolubility of Z. In order to increase the targeting efficiency and/ortherapeutic index using this concept, the second transformation and/orcleavage should only occur after the first transformation and/orcleavage have occurred. If the second transformation and/or cleavage canalso occur before the first transformation and/or cleavage haveoccurred, an improved targeting efficiency and/or an improvedtherapeutic index due to this concept seems unlikely.

It will be apparent that a V² moiety of formula (VI) or a promoietycontaining such a V² cannot only be useful in conjugates of a compoundof formula (I) or (II), but may be used in similar conjugates of othertherapeutic agents, diagnostic moieties, and the like.

A compound of formula (III) containing a V² moiety of formula (VI) maybe prepared from a compound of formula (III) containing a V² moiety offormula (VII):

wherein RM* has the same meaning as RM and is selected independently.

It should be understood that in this document, whenever V², L², L, V¹,Y, RM, p, q, or z is mentioned, the same can apply for each V²*, L²*,L*, V¹*, Y*, RM*, p*, q*, or z*, respectively, unless the contextdictates otherwise.

It should be understood that the functional moiety V² can have severalfunctional properties combined. For example, V² can be a moiety thatimproves the pharmacological properties of a compound of this inventionand at the same time be or contain a targeting moiety.

Conjugates of this invention may contain one or more promoieties. Thesepromoieties may be the same or different. The presence of two or morepromoieties may favorably affect the properties of the conjugate. Forinstance, it may improve the water solubility and/or increase thetargeting efficiency of the conjugate. Furthermore, if in a targetedconjugate there are two promoieties and the promoiety required fortargeting is prematurely cleaved from Z, for example in the circulation,the second promoiety attenuates the cytotoxicity of Z.

In one embodiment, when there are two or more promoieties, saidpromoieties are different from each other. The two or more differentpromoieties may have different functions and may be removed underdifferent conditions and at different extracellular/intracellularlocations.

In one embodiment, there is one promoiety linked to Z. In anotherembodiment, there is one promoiety linked to Z via X¹. In anotherembodiment, there are two promoieties linked to Z. In anotherembodiment, there are two promoieties linked to Z, of which one isconnected via X¹. In another embodiment, there are two promoietieslinked to Z, of which one is connected via X¹ and the other to theDNA-alkylating unit. In another embodiment, there are two promoietieslinked to Z, of which one is connected via X¹ and the other to theDNA-binding unit. In another embodiment, there are two promoietieslinked to Z, of which one is connected to the DNA-binding unit and theother to the DNA-alkylating unit. In yet another embodiment, there arethree promoieties linked to Z. In yet another embodiment, there arethree promoieties linked to Z, of which one is connected via X¹.

In another aspect, this invention relates to conjugates and linker-agentconjugates similar to compounds of formulae (III) and (IV) in which theZ moiety is a therapeutic or diagnostic moiety different from a compoundof formula (I), (II), (I′), or (II′), or a promoiety-containingderivative thereof. Therapeutic moieties may for example be selectedfrom anthracyclines (e.g., daunorubicin, doxorubicin), antimetabolites(e.g., methotrexate, cytarabine, 6-mercaptopurine), calicheamycins,dolastatins, auristatins, tubulysins, epothilones, taxoids (e.g.,paclitaxel, docetaxel), maytansinoids, mitomycins, other alkylatingagents (e.g., melphalan, carmustine, chlorambucil, cyclophosphamide),and other tubulin-binding agents (e.g., vincristine, vinblastine). Allembodiments for compounds of formulae (III) and (IV) also apply toconjugates and linker-agent conjugates similar to compounds of formulae(III) and (IV) in which the Z moiety is a therapeutic or diagnosticmoiety different from a compound of formula (I), (II), (I′), or (II′),or a promoiety-containing derivative thereof, unless the contextdictates otherwise.

In one aspect of this invention, a compound of formula (III) comprisesat least 2 promoieties. The first promoiety contains at least atargeting moiety and the second comprises at least aX¹⁴(CH₂CH₂O)_(gg)CH₂CH₂X¹⁴ moiety or 2X¹⁴CH₂CH₂OCH₂CH₂X¹⁴ moieties, andV^(1′) of said same second promoiety is present. Similarly, a compoundof formula (IV) may comprise at least 2 promoieties. The first promoietycontains at least a reactive moiety RM and the second comprises at leasta X¹⁴(CH₂CH₂O)_(gg)CH₂CH₂X¹⁴ moiety or 2 X¹⁴CH₂CH₂OCH₂CH₂X¹⁴ moieties,and V^(1′) of said same second promoiety is present. Said secondpromoieties of compounds of formulae (III) and (IV) may for example berepresented by

In one embodiment, said second promoiety is selected from

wherein X⁷⁰, X⁷¹, X⁷², and X⁷³ are independently selected from O, S, andNR⁸², X⁷⁴ is selected from

d is selected from 0 to 8, e is 0 or 1, gg′, gg″, gg′″, gg″″, and gg*are independently selected from 0 to 1000, and R⁸¹ and R⁸² areindependently selected from H and optionally substituted C₁₋₃ alkyl. Inother embodiments, gg′, gg″, gg′″, gg″″, and gg* are independentlyselected from 3 to 1000 or 500 or 100 or 50 or 10.

In another embodiment, said second promoiety is selected from

wherein gg′ is selected from 0 to 1000. In other embodiments, gg′ isselected from 3 to 1000 or 500 or 100 or 50 or 10.

In a further embodiment, said second promoiety is selected from

wherein gg′ is selected from 0 to 1000, AS is

wherein A is

R¹¹⁶ is selected from methyl, ethyl, isopropyl, tert-butyl, phenyl,

f is 0, 1, or 2, g is 0 or 1, and PM is an amino acid or a peptidecoupled with its N-terminus to L′.

In other embodiments, gg′ is selected from 3 to 1000 or 500 or 100 or 50or 10 or 5.

In one embodiment, (III) is represented by a compound of formula (III-1)or (III-2):

In more specific embodiments, the DB unit in a compound of formula(III-1) or (III-2) is DB1 or DB2 or DB3 or DB4 or DB5 or DB6 or DB7 orDB8 or DB9.

In another embodiment, a compound of formula (III) is represented by acompound of formula (III-3a) or (III-4a), wherein the DNA-binding moietyis DB1:

wherein Y′ is connected to an atom being part of X³, X³⁴, X⁴, X⁶, X⁷,X⁸, X⁹, X¹¹, or X¹².

In another embodiment, a compound of formula (III) is represented by acompound of formula (III-3b) or (III-4b), wherein the DNA-binding moietyis DB2:

wherein Y′ is connected to an atom being part of X³, X³⁴, X⁴, X⁶, X⁷,X⁹, X¹¹, or X¹².

In another embodiment, a compound of formula (III) is represented by acompound of formula (III-3c) or (III-4c), wherein the DNA-binding moietyis DB3:

wherein Y′ is connected to an atom being part of X⁶, X⁷, X⁸, X⁹, X¹⁰, orX¹¹.

In another embodiment, a compound of formula (III) is represented by acompound of formula (III-3d) or (III-4-d), wherein the DNA-bindingmoiety is DB4:

wherein Y′ is connected to an atom being part of X⁶, X⁷, X⁸, X⁹, or X¹¹.

In another embodiment, a compound of formula (III) is represented by acompound of formula (III-3e) or (III-4-e), wherein the DNA-bindingmoiety is DB5:

wherein Y′ is connected to an atom being part of R^(8b), R^(9b), X³,X³⁴, X⁴, X⁷, or X¹¹.

In another embodiment, a compound of formula (III) is a compound offormula (III-3f) or (III-4f), wherein the DNA-binding moiety is DB6:

wherein Y′ is connected to an atom part of X³, X³⁴, X⁴, X⁶*, X⁷*, X⁸,X⁸*, X⁹, X⁹*, X¹⁰*, or X¹¹*.

In another embodiment, a compound of formula (III) is a compound offormula (III-3g) or (III-4g), wherein the DNA-binding moiety is DB7:

wherein Y′ is connected to an atom being part of X³, X³⁴, X⁴, X⁶*, X⁷,X⁷*, X⁸, X⁸*, X⁹*, or X¹¹*.

In another embodiment, a compound of formula (III) is a compound offormula (III-3h) or (III-4h), wherein the DNA-binding moiety is DB8:

wherein Y′ is connected to an atom being part of X³, X³⁴, X⁴, X⁷, or X⁸.

In another embodiment, a compound of formula (III) is represented by acompound of formula (III-3i) or (III-4i), wherein the DNA-binding moietyis DB9:

wherein Y′ is connected to an atom being part of X⁶, X⁷, X⁸, X⁹, or X¹¹.

This invention further relates to compounds of formulae(III-3j)-(III-3r) and (III-4j)-(III-4r), which are identical tocompounds of formulae (III-3a)-(III-3i) and (III-4a)-(III-4i),respectively, except that the two promoieties have switched places, Ynow being connected to an atom in the DNA-binding unit and Y′ beingconnected to X¹.

It is noted that if in any of compounds of formulae (III-3a)-(III-3i)and (III-4a)-(III-4i) Y′ is connected to a ring atom being part of ringA or ring B instead of to an atom in an R substituent connected to saidring atom, this in fact means that such an R substituent is absent ifthis is necessary to meet valency rules. The same holds for Y incompounds of formulae (III-3j)-(III-3r) and (III-4j)-(III-4r).

In another embodiment, a compound of formula (III) is represented by acompound of formula (III-5) or (III-6):

wherein Y′ is connected to an atom being part of R⁵, R^(5′), R⁶, R^(6′),R⁷, R^(7′), R¹⁴, R^(14′), X² or to any of the atoms bearing these Rsubstituents.

In more specific embodiments, the DB unit in a compound of formula(III-5) or (III-6) is DB1 or DB2 or DB3 or DB4 or DB5 or DB6 or DB7 orDB8 or DB9.

In a further embodiment, a compound of formula (III) is represented bycompounds of formulae (III-7) and (III-8), which are identical tocompounds (III-5) and (III-6), respectively, except that the twopromoieties have switched places, Y now being connected to an atom inthe DNA-alkylating unit and Y′ being connected to X¹.

In more specific embodiments, the DB unit in a compound of formula(III-7) or (III-8) is DB1 or DB2 or DB3 or DB4 or DB5 or DB6 or DB7 orDB8 or DB9.

When Y′ in compounds of formulae (III-5) and (III-6) is connected to aring atom instead of to an atom in an R substituent connected to saidring atom, this in fact means that such an R substituent is absent ifthis is necessary to meet valency rules. The same holds for Y incompounds of formulae (III-7) and (III-8).

Similar embodiments can be envisioned for compounds of formula (IV) byreplacing V²-L² and/or V^(2′)-L^(2′) by RM and/or RM′, respectively, andremoving the parentheses with subscript q and/or q′. Therefore,compounds of formulae (IV-1), (IV-2), (IV-3a)-(IV-3r), (IV-4a)-(IV-4r),(IV-5), (IV-6), (IV-7), and (IV-8) are represented by the structures ofcompounds of formulae (III-1), (III-2), (III-3a)-(III-3r),(III-4a)-(III-4r), (III-5), (III-6), (III-7), and (III-8), respectively,in which at least one of V²-L² and V^(r)-L^(2′) is replaced by RV1 andRM′, respectively.

In one embodiment, the V^(2′)(-L^(2′)-L′(-(V^(1′)—Y′))_(p′))_(q′)(Z′)_(z′-1) moiety in any of compounds of formulae(III-3a)-(III-3r), (III-4a)-(III-4r), (III-5), (III-6), (III-7), and(III-8) or an analogous compound thereof based on a compound of formula(IV) is represented by

In another embodiment, theV^(2′)(-L^(2′)-L′(-(V^(1′)—Y′))_(p′))_(q′)(Z′)_(z′-1) moiety in any ofcompounds of formulae (III-3a)-(III-3r), (III-4a)-(III-4r), (III-5),(III-6), (III-7), and (III-8) or an analogous compound thereof based ona compound of formula (IV) is represented by

In one embodiment, p is an integer from 1 (included) to 128 (included).In another embodiment, q is an integer from 1 (included) to 1000(included). In other embodiments, p is an integer from 1 (included) to64 (included) or 32 (included) or 16 (included) or 8 (included) or 4(included) or 2 (included), or p is 1. In other embodiments, q is aninteger from 1 (included) to 500 (included) or 400 (included) or 300(included) or 200 (included) or 100 (included) or 16 (included) or 8(included) or 6 (included) or 4 (included) or 2 (included), or q is 1.In another embodiment, q is selected from 1 to 4.

In one embodiment, if more than 1 promoiety is connected to a first Zand in one of the promoieties there is more than one attachment site forZ moieties, then the other ones of said promoieties connected to saidfirst Z each contain a single attachment site for a Z moiety.

In one embodiment, a compound of formula (III) is represented by

In one embodiment, p in a compound of formula (IIIa) is 1.

In another embodiment, in a compound of formula (IIIa) p is 1 and zequals q, which reduces formula (IIIa) to:

In another embodiment, a compound of formula (IIIa) is represented by

or by an isomer, or by a mixture of isomers, wherein R⁵, R⁶, R⁷, R¹⁴,and DB are as previously defined, V¹ is selected from valylcitrulline,valyllysine, phenylalanyllysine, alanylphenylalanyllysine, andD-alanylphenylalanyllysine, f is 1 or 2, CL is selected from

L is selected from

q ranges from 1 to 20, rr, rr′, rr″, and rr′″ each independently rangefrom 0 to 8, X⁷⁴ is selected from

each X⁴⁰ and X⁴¹ is independently selected from O, S, and NR¹³⁵, whereinR¹³⁵ is selected from H and C₁₋₃ alkyl, each uu, uu′, and uu″ isindependently selected from 0 and 1, and Ab is an antibody or a fragmentor derivative thereof.

In yet another embodiment, a compound of formula (IIIa) is representedby

or by an isomer, or by a mixture of isomers, wherein R⁵, R⁶, R⁷, R¹⁴,and DB are as previously defined, V¹ is selected from valylcitrulline,valyllysine, phenylalanyllysine, alanylphenylalanyllysine, andD-alanylphenylalanyllysine, f is 1 or 2, CL is

R¹¹⁶ is selected from methyl, ethyl, isopropyl, tert-butyl, phenyl,

L is selected from

q ranges from 1 to 20, rr, rr′, rr″, and rr′″ each independently rangefrom 0 to 8, X⁷⁴ is selected from

each X⁴⁰ and X⁴¹ is independently selected from O, S, and NR¹³⁵, whereinR¹³⁵ is selected from H and C₁₋₃ alkyl, each uu, uu′, and uu″ isindependently selected from 0 and 1, and Ab is an antibody or a fragmentor derivative thereof.

In yet another embodiment, a compound of formula (IIIa) is representedby

or by an isomer, or by a mixture of isomers, wherein R⁵, R⁶, R⁷, R¹⁴,and DB are as previously defined, V¹ is selected from valylcitrulline,valyllysine, phenylalanyllysine, alanylphenylalanyllysine, andD-alanylphenylalanyllysine, f is 1 or 2, CL is selected from

L is selected from

q ranges from 1 to 20, and Ab is an antibody or a fragment or derivativethereof. In yet another embodiment, a compound of formula (IIIa) isrepresented by

or by an isomer, or by a mixture of isomers, wherein R⁵ is selected fromH, methyl and methoxy, R⁶, R⁷, and R¹⁴ are H, DB is DB1, V¹ is selectedfrom valylcitrulline, valyllysine, phenylalanyllysine,alanylphenylalanyllysine, and D-alanylphenylalanyllysine, f is 1 or 2,CL is selected from

L is selected from

q ranges from 1 to 4, and Ab is an antibody or a fragment or derivativethereof.

In another embodiment, a compound of formula (IIIa) is represented by

or by an isomer, or by a mixture of isomers, wherein R⁵, R⁶, R⁷, R¹⁴,and DB are as previously defined, CL and CL′ are independently selectedfrom

V¹ and V^(1′) are independently selected from valylcitrulline,valyllysine, phenylalanyllysine, alanylphenylalanyllysine, andD-alanylphenylalanyllysine, f is 1 or 2, f is 0, 1, or 2, g′ is 0 or 1,the CL′ group—or the p-aminobenzyloxycarbonyl group if g′ is 0, or theV^(1′) group if f is 0 as well—is connected to an atom in DB, L isselected from

q ranges from 1 to 20, rr, rr′, rr″, and rr′″ each independently rangefrom 0 to 8, X⁷⁴ is selected from

each X⁴⁰ and X⁴¹ is independently selected from O, S, and NR¹³⁵, whereinR¹³⁵ is selected from H and C₁₋₃ alkyl, each uu, uu′, and uu″ isindependently selected from 0 and 1, Ab is an antibody or a fragment orderivative thereof, and L′ is selected from

wherein gg′ is selected from 0 to 1000.

In another embodiment, a compound of formula (IIIa) is represented by

or by an isomer, or by a mixture of isomers, wherein R⁵, R⁶, R⁷, R¹⁴,and DB are as previously defined, CL and CL′ are independently selectedfrom

V¹ and V^(1′) are independently selected from valylcitrulline,valyllysine, phenylalanyllysine, alanylphenylalanyllysine, andD-alanylphenylalanyllysine, f is 0, 1, or 2, f is 1, or 2, g is 0 or 1,the CL group—or the p-aminobenzyloxycarbonyl group if g is 0, or the V¹group if f is 0 as well—is connected to an atom in DB, L is selectedfrom

q ranges from 1 to 20, rr, rr′, rr″, and rr′″ each independently rangefrom 0 to 8, X⁷⁴ is selected from

each X⁴⁰ and X⁴¹ is independently selected from O, S, and NR¹³⁵, whereinR¹³⁵ is selected from H and C₁₋₃ alkyl, each uu, uu′, and uu″ isindependently selected from 0 and 1, Ab is an antibody or a fragment orderivative thereof, and L′ is selected from

wherein gg′ is selected from 0 to 1000.

In another embodiment, a compound of formula (III) is represented by

In one embodiment, p* in a compound of formula (IIIa*) is 1.

In another embodiment, in a compound of formula (IIIa*) p* is 1 and z*equals q*.

In another embodiment, in a compound of formula (IIIa*) p* is 1 and z*as well as z equal q*, which reduces formula (IIIa*) to:

In another embodiment, a compound of formula (III) is represented by

In one embodiment, p in a compound of formula (IIIb) is 1.

In another embodiment, p in a compound of formula (IIIb) is 1 and zequals q, which reduces formula (IIIb) to:

In another embodiment, a compound of formula (III) is represented by

In one embodiment, p* in a compound of formula (IIIc) is 1.

In another embodiment, in a compound of formula (IIIc) p* is 1 and z*equals q*.

In yet another embodiment, in a compound of formula (IIIc) p* is 1 andz* as well as z equal q*, which reduces formula (IIIc) to:

In another embodiment, V¹ in a compound of formula (IIIc) is anenzyme-cleavable substrate. In a further embodiment, V¹ can be cleavedby an intracellular enzyme. In another embodiment, V¹ is an optionallysubstituted N,N-dialkylaminocarbonyl group wherein the two alkyl groupsmay be the same or different and optionally be connected to each otherto form an optionally substituted heterocycle. In yet anotherembodiment, V¹ is piperazinocarbonyl. Such a V¹ group may be cleavedenzymatically, for example by carboxylesterases.

In another embodiment, a compound of formula (IIIc) is represented by

or by an isomer, or by a mixture of isomers, wherein R⁵, R⁶, R⁷, R¹⁴,and DB are as previously defined, V¹* is selected from valylcitrulline,valyllysine, phenylalanyllysine, alanylphenylalanyllysine, andD-alanylphenylalanyllysine, P is 1 or 2, L* is selected from

q* ranges from 1 to 20, rr, rr′, rr″, and rr′″ each independently rangefrom 0 to 8, X⁷⁴ is selected from

each X⁴⁰ and X⁴¹ is independently selected from O, S, and NR¹³⁵, whereinR¹³⁵ is selected from H and C₁₋₃ alkyl, each uu, uu′, and uu″ isindependently selected from 0 and 1, and Ab is an antibody or a fragmentor derivative thereof.

In yet another embodiment, a compound of formula (IIIc) is representedby

or by an isomer, or by a mixture of isomers, wherein R⁵, R⁶, R⁷, R¹⁴,and DB are as previously defined, V¹* is selected from valylcitrulline,valyllysine, phenylalanyllysine, alanylphenylalanyllysine, andD-alanylphenylalanyllysine, f* is 1 or 2, L* is selected from

q* ranges from 1 to 20, and Ab is an antibody or a fragment orderivative thereof.

In yet another embodiment, a compound of formula (III) is represented by

V¹—Z  (IIId).

In one embodiment, a compound of formula (IIId) is represented by

or by an isomer, or by a mixture of isomers, wherein R⁵, R⁶, R⁷, R¹⁴,and DB are as previously defined, CL′ is selected from

f′ is 0, 1, or 2, g′ is 0 or 1, V^(1′) is selected from valylcitrulline,valyllysine, phenylalanyllysine, alanylphenylalanyllysine, andD-alanylphenylalanyllysine or is absent, the CL′ group—or thep-aminobenzyloxycarbonyl group if g′ is 0, or the V^(1′) group if f is 0as well, or the L′ group if the V^(1′) group is absent as well—isconnected to an atom in DB, L′ is selected from

q′ ranges from 1 to 20, rr, rr′, rr″, and rr′″ each independently rangefrom 0 to 8, X⁷⁴ is selected from

each X⁴⁰ and X⁴¹ is independently selected from O, S, and NR¹³⁵, whereinR¹³⁵ is selected from H and C₁₋₃ alkyl, each uu, uu′, and uu″ isindependently selected from 0 and 1, Ab is an antibody or a fragment orderivative thereof, and V¹ is selected from a mono-, di-, oroligosaccharide or a reduced, oxidized, or protected derivative thereofand

wherein R¹⁴¹, R¹⁴², and R¹⁴³ are independently selected from H andoptionally substituted C₁₋₈ alkyl, C₁₋₈ heteroalkyl, C₃₋₈ cycloalkyl,C₁₋₈ heterocycloalkyl, C₅₋₈ aryl, or C₁₋₈ heteroaryl.

In another embodiment, a compound of formula (IIId) is represented by

or by an isomer, or by a mixture of isomers, wherein R⁵, R⁶, R⁷, R¹⁴,and DB are as previously defined, CL′ is

R¹¹⁶ is selected from methyl, ethyl, isopropyl, tert-butyl, phenyl,

f′ is 0, 1, or 2, g′ is 0 or 1, V^(1′) is selected from valylcitrulline,valyllysine, phenylalanyllysine, alanylphenylalanyllysine, andD-alanylphenylalanyllysine or is absent, the CL′ group—or thep-aminobenzyloxycarbonyl group if g′ is 0, or the V^(1′) group if f′ is0 as well, or the L′ group if the V^(1′) group is absent as well—isconnected to an atom in DB, L′ is selected from

q′ ranges from 1 to 20, rr, rr′, rr″, and rr′″ each independently rangefrom 0 to 8, X⁷⁴ is selected from

each X⁴⁰ and X⁴¹ is independently selected from O, S, and NR¹³⁵, whereinR¹³⁵ is selected from H and C₁₋₃ alkyl, each uu, uu′, and uu″ isindependently selected from 0 and 1, Ab is an antibody or a fragment orderivative thereof, and V¹ is selected from a mono-, di-, oroligosaccharide or a reduced, oxidized, or protected derivative thereofand

wherein R¹⁴¹, R¹⁴², and R¹⁴³ are independently selected from H andoptionally substituted C₁₋₈ alkyl, C₁₋₈ heteroalkyl, C₃₋₈ cycloalkyl,C₁₋₈ heterocycloalkyl, C₅₋₈ aryl, or C₁₋₈ heteroaryl.

In another embodiment, a compound of formula (IIId) is represented by

or by an isomer, or by a mixture of isomers, wherein R⁵, R⁶, R⁷, R¹⁴,and DB are as previously defined, CL′ is selected from

f′ is 1 or 2, V^(1′) is selected from valylcitrulline, valyllysine,phenylalanyllysine, alanylphenylalanyllysine, andD-alanylphenylalanyllysine, L′ is selected from

q′ ranges from 1 to 20, rr, rr′, rr″, and rr′″ each independently rangefrom 0 to 8, each X⁴⁰ and X⁴¹ is independently selected from O, S, andNR¹³⁵, wherein R¹³⁵ is selected from H and C₁₋₃ alkyl, each uu, uu′, anduu″ is independently selected from 0 and 1, Ab is an antibody or afragment or derivative thereof, and V¹ is coupled to an atom of DB andis selected from a mono-, di-, or oligosaccharide or a reduced,oxidized, or protected derivative thereof and

wherein R¹⁴¹, R¹⁴², and R¹⁴³ are independently selected from H andoptionally substituted C₁₋₈ alkyl, C₁₋₈ heteroalkyl, C₃₋₈ cycloalkyl,C₁₋₈ heterocycloalkyl, C₅₋₈ aryl, or C₁₋₈ heteroaryl.

In another embodiment, a compound of formula (IIId) is represented by

or by an isomer, or by a mixture of isomers, wherein R⁵, R⁶, R⁷, R¹⁴,and DB are as previously defined, CL′ is

R¹¹⁶ is selected from methyl, ethyl, isopropyl, tert-butyl, phenyl,

f′ is 1 or 2, V^(1′) is selected from valylcitrulline, valyllysine,phenylalanyllysine, alanylphenylalanyllysine, andD-alanylphenylalanyllysine, L′ is selected from

q′ ranges from 1 to 20, rr, rr′, rr″, and rr′″ each independently rangefrom 0 to 8, each X⁴⁰ and X⁴¹ is independently selected from O, S, andNR¹³⁵, wherein R¹³⁵ is selected from H and C₁₋₃ alkyl, each uu, uu′, anduu″ is independently selected from 0 and 1, Ab is an antibody or afragment or derivative thereof, and V¹ is coupled to an atom of DB andis selected from a mono-, di-, or oligosaccharide or a reduced,oxidized, or protected derivative thereof and

wherein R¹⁴¹, R¹⁴², and R¹⁴³ are independently selected from H andoptionally substituted C₁₋₈ alkyl, C₁₋₈ heteroalkyl, C₃₋₈ cycloalkyl,C₁₋₈ heterocycloalkyl, C₅₋₈ aryl, or C₁₋₈ heteroaryl.

In yet another embodiment, a compound of formula (III) is represented by

V²-L²-L-Z  (IIIe).

Similar embodiments can be envisioned for compounds of formula (IV) byreplacing V²-L² and/or V^(2′)-L^(2′) by RM and/or RM′, respectively, andremoving the parentheses with subscript q and/or q′, or by eliminatingAb-SH from the structures. Therefore, compounds of formulae (IVa)-(IVe)are represented by the structures of compounds of formulae(IIIa)-(IIIe), respectively, in which V²-L² or V^(2′)-L^(2′) is replacedby RM or RM′, respectively, and parentheses with subscript q or q′ areremoved.

In one embodiment, the DB moiety of a Z in a compound of formula (III)is DB1.

In other embodiments, the DB moiety of a Z in a compound of formula(III) is DB2 or DB3 or DB4 or DB5 or DB6 or DB7 or DB8 or DB9.

In another embodiment, the DB moiety of a Z in a compound of formula(IV) is DB1.

In other embodiments, the DB moiety of a Z in a compound of formula (IV)is DB2 or DB3 or DB4 or DB5 or DB6 or DB7 or DB8 or DB9.

In yet another embodiment, the DB moiety of a Z in a compound of formula(III) is

In yet another embodiment, the DB moiety of a Z in a compound of formula(IV) is

In one embodiment, a compound of formula (IV) is represented by

or by an isomer, or by a mixture of isomers, wherein R^(5a) is selectedfrom H, methyl and methoxy, AZ is

V¹ is selected from valylcitrulline, valyllysine, phenylalanyllysine,alanylphenylalanyllysine, and D-alanylphenylalanyllysine, CL is

R¹¹⁶ is selected from methyl, ethyl, isopropyl, tert-butyl, phenyl,

and L is selected from

In another embodiment, a compound of formula (IV) is represented by

or by an isomer, or by a mixture of isomers, wherein R^(5a) is selectedfrom H, methyl and methoxy, AZ is

V¹ is selected from valylcitrulline, valyllysine, phenylalanyllysine,alanylphenylalanyllysine, and D-alanylphenylalanyllysine, CL is

and L is selected from

In another embodiment, a compound of formula (III) is represented by

or by an isomer, or by a mixture of isomers, wherein R^(5a) is selectedfrom H, methyl and methoxy, AZ is

V¹ is selected from valylcitrulline, valyllysine, phenylalanyllysine,alanylphenylalanyllysine, and D-alanylphenylalanyllysine, CL is

R¹¹⁶ is selected from methyl, ethyl, isopropyl, tert-butyl, phenyl,

q ranges from 1 to 8, Ab is an antibody or a fragment or derivativethereof, and L is selected from

In yet another embodiment, a compound of formula (III) is represented by

or by an isomer, or by a mixture of isomers, wherein R^(5a) is selectedfrom H, methyl and methoxy, AZ is

V¹ is selected from valylcitrulline, valyllysine, phenylalanyllysine,alanylphenylalanyllysine, and D-alanylphenylalanyllysine, CL is

L is selected from

q ranges from 1 to 4, and Ab is an antibody or a fragment or derivativethereof.

A series of compounds (III-AZ) wherein R^(5a) is methyl, AZ is

L is

Ab is Trastuzumab, V¹ is valylcitrulline, and CL is

were prepared according to the procedure described in Example 15 andevaluated against the corresponding reference compound in which CL is

for amount of aggregate present after the conjugation step. Theevaluated compounds had relative aggregate amounts of 33%, 15%, 8%, 58%,and 12%, respectively, compared to that of the reference compound(100%), clearly showing the advantage of the present cyclizationspacers.

In one embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

In another embodiment, a compound of formula (IV) is selected from

Synthesis of Compounds of the Invention

Compounds of formulae (I)-(IV) and (VIII) can be conveniently preparedin a way for some part analogous to compounds reported in WO 01/83448,WO 02/083180, WO 2004/043493, WO 2007/018431, WO 2007/089149, WO2009/017394, and WO2010/062171. Further representative syntheticprotocols can be found in the Examples.

In one embodiment, a compound of formula (I) or (II) is used to preparea compound of formula (III). In another embodiment, a compound offormula (I) or (II) is used to prepare a compound of formula (IV). Inanother embodiment, a compound of formula (IV) is used to prepare acompound of formula (III). In another embodiment, a compound of formula(III) wherein V¹ is a protecting group is used to prepare anothercompound of formula (III) wherein V¹ is an in vivocleavable/transformable moiety. In yet another embodiment, a compound offormula (VIII) is used to prepare a compound of formula (IV). In yetanother embodiment, a compound of formula (VIII) is used to prepare acompound of formula (III), optionally via a compound of formula (IV).

Uses, Methods, and Compositions

In one aspect, this invention relates to use of a compound of formula(I) or (II) for the preparation of a compound of formula (III).

In another aspect, this invention relates to use of a compound offormula (IV) for the preparation of a compound of formula (III).

In yet another aspect, this invention relates to use of a compound offormula (I) or (II) for the preparation of a compound of formula (IV).

In yet another aspect, this invention relates to use of a compound offormula (VIII) for the preparation of a compound of formula (IV).

In yet another aspect, this invention relates to use of a compound offormula (VIII) for the preparation of a compound of formula (III),optionally via a compound of formula (III).

In yet another aspect, this invention relates to use of a compound offormula (VIII) for the preparation of conjugates and linker-agentconjugates similar to compounds of formulae (III) and (IV) in which theZ moiety is a therapeutic or diagnostic moiety different from a compoundof formula (I), (II), (I′), or (II′), or a promoiety-containingderivative thereof.

In yet another aspect, this invention relates to use of a compound offormula (III) wherein V¹ is a protecting group for the preparation ofanother compound of formula (III) wherein V¹ is an in vivocleavable/transformable moiety.

In yet another aspect, the invention relates to the use of any of thecompounds defined hereinabove for the manufacture of a pharmaceuticalcomposition for the treatment of a mammal being in need thereof. In oneembodiment, the invention relates to the use of any of the compoundsdefined hereinabove for the manufacture of a pharmaceutical compositionfor the treatment of a tumor in a mammal. In another embodiment, theinvention relates to the use of any of the compounds defined hereinabovefor the manufacture of a pharmaceutical composition for the preventionof a tumor in a mammal. The invention can also be worded as any of thecompounds defined hereinabove for use in the treatment of a mammal beingin need thereof, or for use in the treatment of a tumor in a mammal orfor use in the prevention of a tumor in a mammal.

The invention also relates to any of the compounds defined hereinaboveas a medicament or an active component or active substance in amedicament.

In a further aspect, the invention relates to a process for preparing apharmaceutical composition containing a compound as defined hereinabove,to provide a solid or a liquid formulation for administration orally,topically, or by injection. Such a method or process at least comprisesthe step of mixing the compound with a pharmaceutically acceptablecarrier.

In one embodiment, a compound of the invention is used to treat orprevent an illness characterized by undesired proliferation. In anotherembodiment, a compound of the invention is used to treat or prevent anillness characterized by undesired cell proliferation. In anotherembodiment, a compound of the invention is used to treat a tumor. Inanother embodiment, a compound of the invention is used to prevent atumor. In yet another embodiment, a compound of the invention is used totreat or prevent an inflammatory disease. In yet another embodiment, acompound of the invention is used to treat or prevent an autoimmunedisease. In yet another embodiment, a compound of the invention is usedto treat or prevent a bacterial, viral, or microbial infection.

In a further embodiment, this invention relates to a method of treatinga mammal having an illness characterized by undesired (cell)proliferation with a compound of this invention. In another embodiment,this invention relates to a method of treating a mammal carrying a tumorwith a compound of this invention. In yet another embodiment, thisinvention relates to a method of treating a mammal having aninflammatory disease with a compound of this invention. In yet anotherembodiment, this invention relates to a method of treating a mammalhaving an autoimmune disease with a compound of this invention. In yetanother embodiment, this invention relates to a method of treating amammal having a bacterial, viral, or microbial infection with a compoundof this invention.

In a further embodiment, the invention relates to a method of treating amammal being in need thereof, whereby the method comprises theadministration of a pharmaceutical composition comprising a compound ofthis invention to the mammal in a therapeutically effective dose.

In one embodiment, the invention relates to a method of treating orpreventing a tumor in a mammal, whereby the method comprises theadministration of a pharmaceutical composition comprising a compound ofthis invention to the mammal in a therapeutically effective dose. Inanother embodiment, the invention relates to a method of treating atumor in a mammal, whereby the method comprises the administration of apharmaceutical composition comprising a compound of this invention tothe mammal in a therapeutically effective dose. In yet anotherembodiment, the invention relates to a method of treating a tumor in ahuman, whereby the method comprises the administration of apharmaceutical composition comprising a compound of this invention tothe human in a therapeutically effective dose.

In another embodiment, the invention relates to a method of treating orpreventing an inflammatory disease in a mammal, whereby the methodcomprises the administration of a pharmaceutical composition comprisinga compound of this invention to the mammal in a therapeuticallyeffective dose.

In another embodiment, the invention relates to a method of treating orpreventing an autoimmune disease in a mammal, whereby the methodcomprises the administration of a pharmaceutical composition comprisinga compound of this invention to the mammal in a therapeuticallyeffective dose.

In another embodiment, the invention relates to a method of treating orpreventing a bacterial, viral, or microbial infection in a mammal,whereby the method comprises the administration of a pharmaceuticalcomposition comprising a compound of this invention to the mammal in atherapeutically effective dose.

The invention also relates to pharmaceutical compositions comprising thecompounds of the invention as defined hereinabove. A compound of theinvention may be administered in purified form together with apharmaceutical carrier as a pharmaceutical composition. The preferredform depends on the intended mode of administration and therapeuticapplication. The pharmaceutical carrier can be any compatible, nontoxicsubstance suitable to deliver the compounds of the invention to thepatient. Pharmaceutically acceptable carriers are well known in the artand include, for example, aqueous solutions such as (sterile) water orphysiologically buffered saline or other solvents or vehicles such asglycols, glycerol, oils such as olive oil or injectable organic esters,alcohol, fats, waxes, and inert solids. A pharmaceutically acceptablecarrier may further contain physiologically acceptable compounds thatact for example to stabilize or to increase the absorption of thecompounds of the invention. Such physiologically acceptable compoundsinclude, for example, carbohydrates, such as glucose, sucrose, ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins, or other stabilizers orexcipients. One skilled in the art would know that the choice of apharmaceutically acceptable carrier, including a physiologicallyacceptable compound, depends, for example, on the route ofadministration of the composition. Pharmaceutically acceptableadjuvants, buffering agents, dispersing agents, and the like, may alsobe incorporated into the pharmaceutical compositions.

For oral administration, the active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. Activecomponent(s) can be encapsulated in gelatin capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonate,and the like. Examples of additional inactive ingredients that may beadded to provide desirable color, taste, stability, buffering capacity,dispersion, or other known desirable features are red iron oxide, silicagel, sodium lauryl sulfate, titanium dioxide, edible white ink, and thelike. Similar diluents can be used to make compressed tablets. Bothtablets and capsules can be manufactured as sustained release productsto provide for continuous release of medication over a period of hours.Compressed tablets can be sugar-coated or film-coated to mask anyunpleasant taste and protect the tablet from the atmosphere, orenteric-coated for selective disintegration in the gastrointestinaltract. Liquid dosage forms for oral administration can contain coloringand flavoring to increase patient acceptance.

The compounds of the invention are however preferably administeredparenterally. Preparations of the compounds of the invention forparenteral administration must be sterile. Sterilization is readilyaccomplished by filtration through sterile filtration membranes,optionally prior to or following lyophilization and reconstitution. Theparenteral route for administration of compounds of the invention is inaccord with known methods, e.g. injection or infusion by intravenous,intraperitoneal, intramuscular, intraarterial, or intralesional routes.The compounds of the invention may be administered continuously byinfusion or by bolus injection. A typical composition for intravenousinfusion could be made up to contain 100 to 500 ml of sterile 0.9% NaClor 5% glucose optionally supplemented with a 20% albumin solution and 1mg to 10 g of the compound of the invention, depending on the particulartype of compound of the invention and its required dosing regime.Methods for preparing parenterally administrable compositions are wellknown in the art and described in more detail in various sources,including, for example, Remington's Pharmaceutical Science ¹⁷.

A compound of the invention may also be used in combination therapy, inwhich a compound of this invention is used in combination with one ormore other therapeutic agents. Combination of two or more therapeuticsmay favorably affect treatment outcome. The agents may be administeredeither sequentially or concomitantly. Therefore, in one embodiment thisinvention relates to use of a compound of this invention or apharmaceutical composition comprising a compound of this invention incombination therapy.

The invention is further exemplified by the following examples. Theseexamples are for illustrative purposes only and are not intended tolimit the scope of the invention.

EXAMPLES Example 1 Synthesis of Boc-Protected Cyclization Spacers

Route A

Synthesis of Compound 1:

7.5 ml (85 mmol) oxalyl chloride was dissolved in 200 ml DCM and cooledto T<−60° C. and 12.1 ml (171 mmol) DMSO in 10 ml DCM was added dropwise(T<−60° C.) and stirred an additional 10 min. 10.0 g (57 mmol)N-Boc-N-methylaminoethanol in 40 ml DCM were added dropwise (T<−60° C.)and stirred an additional 10 min. 40 ml (285 mmol) Et₃N was addeddropwise followed by 50 ml DCM (T<−60° C.) and stirred for 30 min. Thereaction mixture was warmed to 0° C. and washed with 3×100 ml water, 100ml 0.5 M KHSO₄, 75 ml brine, dried with MgSO₄ and concentrated in vacuo.The product was purified by column chromatography (SiO₂, DCM/ethylacetate, 1:0 to 9:1) to give 7.36 g (74%) of compound 1. ¹H-NMR (300MHz, CDCl₃): δ=1.42/1.46 (s, 9H, Boc), 2.93/2.96 (s, 3H, Me), 3.90/4.01(s, 2H, CH₂), 9.60 (s, 1H, CHO). Z/E isomers.

General Procedure Reductive Amination:

1 mmol R-amine and 1 mmol compound 1 were stirred in 10 ml MeOH for 4hrs. The reaction mixture was cooled in ice and 2 mmol sodiumborohydride was added in portions and the mixture stirred at RTovernight. The mixture was concentrated and purified by columnchromatography (SiO₂, DCM/MeOH, 1:0 to 1:1) to giveN-Boc-N-Methyl-N′—R-diaminoethane.

Compound 4a, R═(CH₂CH₂O)₂H:

40% yield, ¹H NMR (300 MHz, CDCl₃): δ=1.46 (9H, s, Boc), 2.75-2.87 (4H,m, 2×CH₂), 2.88 (3H, s, NMe), 3.35 (2H, t, J=6.6 Hz, CH₂), 3.56-3.63(4H, m, 2×CH₂, CH), 3.70-3.75 (2H, m, CH₂, CH); MS (ESI) m/z=263.5(M+H⁺).

Compound 4b, R═(CH₂)₃COOMe:

61% yield, ¹H NMR (300 MHz, CDCl₃): δ=1.46 (9H, s, Boc), 1.83 (2H, m,CH₂), 2.39 (2H, t, J=7.4 Hz, CH₂) 2.64-2.86 (3H, m, CH/CH₂), 2.88 (3H,s, NMe), 2.99 (1H, m, CH), 3.30-3.49 (2H, m, CH₂), 3.67 (3H, s, CH₃); MS(ESI) m/z=275.5 (M+H⁺).

Compound 4c:

¹H-NMR (300 MHz, DMSO-d₆): δ=1.15 (3H, d, J=6.9 Hz, CH₃ Ala), 1.38 (9H,s, Boc), 2.57-2.67 (1H, m, α-H), 2.76 (3H, s, NCH₃), 3.08-3.36 (4H, m,CH₂—CH₂), 3.63 (3H, s, OCH₃); MS (ESI): m/z=261.3 (M+H⁺).

Compound 4d:

¹H-NMR (300 MHz, DMSO-d₆): δ=1.12 (3H, d, J=6.9 Hz, CH₃ Ala), 1.39 (9H,s, Boc), 2.54-2.61 (1H, m, α-H), 2.77 (3H, s, NCH₃), 3.10-3.51 (13H, m,6×CH₂+OH), 7.90 (1H, s, NH amide); MS (ESI): m/z=334.4 (M+H⁺).

Compound 4e:

27% yield, ¹H-NMR (300 MHz, CDCl₃): δ=1.44 (9H, s, Boc), 2.72-2.85 (4H,m, 2×CH₂), 2.81 (3H, s, MeN), 3.31 (2H, m, NCH₂), 3.5-3.7 (32H, m,15×CH₂+OH+NH); MS (ESI): m/z=527.4 (M+H⁺).

Compound 4f:

30% yield, ¹H-NMR (300 MHz, CDCl₃): δ=1.44 (9H, s, Boc), 2.65-2.79 (4H,m, 2×CH₂), 2.80 (3H, s, MeN), 3.26 (2H, m, NCH₂), 3.5-3.7 (48H, m,23×CH₂+OH+NH); MS (ESI): m/z 703.5 (M+H⁺).

Compound 4g:

¹H-NMR (300 MHz, DMSO-d₆): δ=1.14 (5H, m, 2 CH and CH₃), 1.35 (9H, s,Boc), 1.47 (4H, m, 4 CH), 1.74-2.38 (4H, m, 4 CH), 2.59 (2H, m, CH₂),2.75 (3H, s, NCH₃), 3.16 (2H, m, CH₂), 3.99 (2H, m, CH₂); MS (ESI):m/z=329.4 (M+H⁺).

Compound 4h:

63% yield, ¹H-NMR (300 MHz, CDCl₃): δ=1.46 (9H, s, Boc), 1.69 (2H, m,CH₂), 2.24 (6H, s, Me₂), 2.36 (2H, t, CH₂), 2.52 (1H, m, NH), 2.72 (2H,t, CH₂), 2.80 (2H, t, CH₂), 2.88 (3H, s, MeN), 3.37 (2H, m, CH₂); MS(ESI) m/z=260.2 (M+H⁺).

Compound 4i:

¹H-NMR (300 MHz, DMSO-d₆): δ=1.30-1.36 (4H, m, 2 CH₂ Lys), 1.35 (18H, 2s, 2 Boc), 1.53 (2H, m, CH₂ Lys), 2.46 (2H, m, CH₂ Lys), 2.56 (2H, m,CH₂), 2.75 (3H, s, NCH₃), 3.13 (3H, m, CH₂ and α-H Lys), 3.58 (3H, s,OCH₃); MS (ESI): m/z=481.4 (M+H⁺).

Compound 4j:

¹H-NMR (300 MHz, DMSO-d₆): δ=1.36 (31H, m, 1 tBu, 2 Boc and 2 CH₂ Lys),1.53 (2H, m, CH₂ Lys), 2.46 (2H, m, CH₂ Lys), 2.56 (2H, t, CH₂), 2.75(3H, s, NCH₃), 3.14 (2H, t, CH₂) 3.64 (1H, m, α-H Lys), 7.01 (d, 1H,NH); MS (ESI): m/z=460.3 (M+H⁺).

Compound 4k:

¹H NMR (300 MHz, CDCl₃): δ=1.15 (2H, m), 1.30 (2H, m), 1.94 (4H, m),2.46 (1H, m), 2.76 (2H, t, J=6.8 Hz), 2.87 (3H, s), 3.30 (2H, t, J=6.8Hz), 3.60 (1H, m); MS (ESI): m/z=273.3 (M+H⁺).

Route B

General Procedure Alkylation Reaction:

To a solution of 1 mmol R-amine and 1 mmol mesylate in dry THF (1.5 mL)was added K₂CO₃ (1.5 mmol) and the mixture was stirred at 60° C.overnight. The mixture was cooled to RT, concentrated and purified bycolumn chromatography (SiO₂, DCM/MeOH, 1:0 to 1:1) to give theN-Boc-N-Methyl-N′—R-diaminoethane.

Compound 5a, R=iso-propyl:

¹H NMR (300 MHz, CDCl₃), δ (ppm): 1.05 (6H, d, 2×CH₃), 1.46 (9H, s,Boc), 2.75 (2H, t, J=6.6 Hz, CH₂), 2.82 (1H, t, J=6.6 Hz, CH), 2.87 (3H,s, NMe), 3.31 (2H, t, J=6.6 Hz, CH₂); MS (ESI) m/z=217.2 (M+H⁺).

Compound 5b:

Starting from N-Boc-prolinol¹⁸ compound 5b was prepared according toroute B. MS (ESI) m/z=215.2 (M+H⁺).

Compound 5c:

¹H-NMR (300 MHz, DMSO-d₆): δ=1.39 (9H, s, Boc), 2.73-2.85 (7H, m,NCH₃+2×CH₂), 3.24-3.30 (2H, m, CH₂), 3.39-3.55 (11H, m, 5×CH₂+OH).

Example 2 Synthesis of Compound 2

2.28 g (10 mmol) benzyl phenylcarbonate was dissolved in 20 ml ethanoland 1.03 ml (10 mmol) 1,2-diamino-1-methylpropane was added dropwise andstirred at RT overnight. The mixture was diluted with 25 ml water andacidified with 1M HCl until pH<3 and extracted with DCM. The aqueousphase was basified with 4M NaOH and extracted with DCM. The extract wasdried with MgSO₄ and concentrated in vacuo. This gave 1.93 g (87%)monoprotected diamine. This material was dissolved in 25 ml dioxane,2.028 g (10.5 mmol) Boc₂O and 0.12 g (1 mmol) DMAP were added and thereaction was stirred at RT overnight. The mixture was concentrated invacuo and purified by column chromatography (SiO₂, ether/heptane, 1:0 to7:3) to give 1.13 g (40%) of diprotected diamine. The diprotecteddiamine was dissolved in 10 ml dry DMF, 1.1 ml (17.5 mmol) iodomethanewas added and the reaction mixture cooled in ice. 0.50 g (10.5 mmol)sodium hydride (60% in oil) was added in portions and stirred in ice for2 hrs. The mixture was warmed to RT, quenched with 10 ml saturated NH₄Cland 50 ml water, extracted with ethyl acetate, dried with MgSO₄ andconcentrated in vacuo. The product was purified by column chromatography(SiO₂, DCM/ethyl acetate, 1:0 to 50:1) to give 0.358 g (29%) ofCbz-protected compound 2. ¹H-NMR (300 MHz, CDCl₃): δ=1.29 (s, 3H, Me),1.35 (s, 3H, Me), 1.46 (s, 9H, Boc), 2.82/2.86 (s, 3H, Me, Z/E), 2.93(s, 3H, Me), 3.72 (s, 2H, CH₂N), 5.12 (s, 2H, benzyl), 7.35 (m, 5H,Phe). This material was dissolved in 20 ml methanol, 0.04 g Pd/C wasadded, the mixture was stirred under hydrogen for 3 hrs, filtered, andthe filtrate concentrated. This gave 0.21 g (100%) of compound 2. MS(ESI): m/z=217.2 (M+H⁺).

Example 3 Synthesis of Compound 3

2.15 g (18.8 mmol) cis-1,2-diaminocyclohexane was dissolved in 20 mlethyl formate and refluxed overnight. The suspension was cooled,filtered and dried under vacuum to give 2.59 g (80%) of product. Thesolid was added in portions to a cooled mixture of 2.1 g (53 mmol)lithium aluminumhydride in 50 ml THF and the mixture was stirred at RTfor 1 hr and then refluxed overnight. The clear solution was cooled inice and quenched by dropwise addition of 2.1 ml water followed by 15 ml20% NaOH. The suspension was briefly heated until the salts were white.The mixture was cooled to RT and filtered. The residue was washed with 2portions of 50 ml THF. The filtrate was concentrated and dissolved in 50ml DCM and washed with 20 ml 4M NaOH. The aqueous phase was extractedwith 25 ml DCM, dried with MgSO₄ and concentrated in vacuo to give 1.94g (89%) of product. The crude product was dissolved in 50 ml DCM and themixture cooled to −40° C. and 2.95 g (13.5 mmol) Boc₂O in 10 ml DCM wasadded dropwise. The mixture was slowly warmed to RT and washed with 20ml 2M NaOH, dried with MgSO₄, concentrated in vacuo and the crudeproduct purified by column chromatography (SiO₂, DCM/MeOH, 1:0 to 9:1)to give 2.09 g (63%) of compound 3. ¹H-NMR (300 MHz, CDCl₃): δ=1.38 (m,6H, cyclohex), 1.46 (s, 9H, Boc), 1.83 (m, 3H, cyclohex+NH), 2.35 (s,3H, NMe), 2.89 (s, 3H, NMe), 2.95 (m, 1H, CHN), 3.86 (m, 1H, CHN); MS(ESI): m/z=243.2 (M+H⁺).

Example 4 Synthesis of Compound 6

To a solution of 2.55 mmol N-Boc-prolinal in dry THF (5 mL) under anArgon atmosphere was added 0.7 mL glacial acetic acid and 272 mg2-(2-aminoethoxy)ethanol (2.58 mmol). The mixture was stirred overnightat room temperature. 2.59 mmol sodium borohydride was added portionwise, and the mixture was stirred for another 4 hrs after which waterwas added. The mixture was washed with ethyl acetate, the aqueous layerbasified up to pH 10 using Na₂CO₃ and extracted with ethyl acetate (9×).The combined organic layers were washed with brine, dried with MgSO₄,and concentrated in vacuo to give 335 mg (46%) crude 6, which was usedwithout further purification. MS (ESI): m/z=289 (M+H⁺).

Example 5 Synthesis of Compounds 7 and 8

0.17 g (1.5 mmol) N-methylpiperid-4-one and 0.178 g (1.0 mmol)N-Boc-N-methyl-1,2-diaminoethane were reacted with 0.42 g (2.0 mmol)sodium triacetoxyborohydride in 5 ml DCE at RT overnight. The reactionwas quenched with MeOH and the mixture extracted with DCM and brine(basified to pH>12), dried with MgSO₄ and concentrated in vacuo to give0.31 g (100%) of 7 as an oil. ¹H-NMR (300 MHz, CDCl₃): δ=¹H-NMR (300MHz, CDCl₃): δ=1.38 (2, m, CH₂), 1.45 (9H, s, Boc), 1.76 (1H, br s, NH),1.85 (2H, m, CH₂), 2.00 (2H, m, CH₂), 2.27 (3H, s, MeN), 2.46 (1H, m,CH), 2.76 (4H, m, 2×CH₂), 2.86 (3H, s, MeN), 3.30 (2H, t, CH₂); MS (ESI)m/z=272.2 (M+H⁺).

Compound 8 was prepared similarly to compound 7 using 0.75 g (4.0 mmol)N-hydroxyethoxyethyl-4-piperidine. The crude product was purified bycolumn chromatography (SiO₂, DCM/MeOH, 1:0 to 95:5) to give 0.83 g (66%)of 8 as an oil. ¹H-NMR (300 MHz, CDCl₃): δ=1.46 (9H, s, Boc), 1.89 (2H,m, CH₂), 1.91 (2H, m, CH₂), 2.50 (3H, m, CH+OH+NH), 2.57 (2H, t, CH₂),2.77 (2H, t, CH₂), 2.87 (3H, s, MeN), 2.94 (2H, m, CH₂), 3.30 (2H, t,CH₂), 3.64 (6H, m, 3×CH₂O); MS (ESI) m/z=346.3 (M+H⁺).

Example 6 Synthesis of Compound 9

0.18 g (1.0 mmol) N-Boc-N-methyl-1,2-diaminoethane and 0.15 g (1.0 mmol)4-formylbenzoic acid were reacted according to general procedure A ofexample 1 to give 0.34 g (90%) 9 (HCl salt). ¹H-NMR (300 MHz, DMSO-d₆):δ=2.89 (3H, s, MeN), 3.11 (2H, m, CH₂N), 3.57 (2H, t, CH₂N), 4.25 (2H,m, CH₂N), 7.33 (2H, d, ArH), 8.02 (2H, d, ArH), 9.03 (2H, br s, NH₂+),13.03 (1H, s, CO₂H); MS (ESI) m/z=309.2 (M+H⁺).

Example 7 Synthesis of Compound 11

1.84 g (7.5 mmol) N-Cbz-1,3-diamine hydrochloride and 3 ml Et₃N weredissolved in 50 ml DCM, cooled in ice and 0.94 ml phenyl chloroformatedissolved in 5 ml DCM was added dropwise. The mixture was stirred at 0°C. for 2 hrs and concentrated in vacuo. The residue was dissolved in 50ml DCM, washed with 25 ml 0.5 M KHSO₄, dried with MgSO₄ and concentratedin vacuo. The residue was dissolved in 25 ml EtOH, 4 ml concentratedammonia was added and the mixture was stirred for 3 hrs. The mixture wasconcentrated in vacuo and the residue dissolved in 25 ml CHCl₃, washedwith 10 ml 4 M NaOH and brine, dried with MgSO₄ and concentrated invacuo to give 1.9 g of intermediate. This material was dissolved in 50ml MeOH, 0.1 g Pd/C was added and the reaction mixture stirred underhydrogen for 5 hrs. The mixture was filtered and the filtrateconcentrated in vacuo to give 0.87 g (95%) of compound 10. 0.26 g (1.7mmol) of this amine was reacted according to general procedure A to give0.17 g (36%) of 11 as an oil. ¹H-NMR (300 MHz, DMSO-d₆): δ=1.39 (9H, s,Boc), 1.45 (2H, m, CH₂), 2.48 (2H, t, CH₂), 2.58 (2H, t, CH₂), 2.78 (3H,s, MeN), 2.97 (2H, m, CH₂), 3.18 (2H, t, CH₂), 5.35 (2H, s, NH₂), 5.87(1H, t, NH); MS (ESI) m/z=275.2 (M+H⁺).

Example 8 Synthesis of Compound 12

0.98 g (4.0 mmol) N-Cbz-1,3-diamine hydrochloride and 3 ml Et₃N weredissolved in 15 ml DCM and added dropwise to 1.65 ml (12 mmol) diethyloxalate in 15 ml ethanol. The mixture was stirred at RT overnight,concentrated in vacuo and the residue purified by column chromatography(SiO₂, DCM/MeOH, 1:0 to 95:5) to give the mono-oxalamide. This materialwas dissolved in 15 ml ethanol and 5 ml concentrated ammonia was addedand the mixture was stirred at RT overnight. The mixture was dilutedwith 50 ml MeOH and 100 mg Pd/C was added. The mixture was stirred underhydrogen atmosphere for 3 hrs. 10 ml water was added, the mixture wasfiltered over Celite and the filtrate concentrated in vacuo to give 0.57g (94%) of white solid. This was reacted according to general procedureA of Example 1 to give 0.45 g (38%) of 12 as a white solid. ¹H-NMR (300MHz, CDCl₃): δ=1.45 (9H, s, Boc), 1.80 (2H, m, CH₂), 2.80 (4H, m,2×CH₂), 2.89 (3H, s, MeN), 3.39 (4H, m, 2×CH₂), 6.85 (1H, s, NH), 7.40(1H, s, NH), 8.39 (1H, t, NH); MS (ESI) m/z=303.2 (M+H⁺).

Example 9 Synthesis of Compounds 14a-b and 15a-b

Compound 14c:

Compound 13c (0.51 mmol) was dissolved in THF (12 ml); LiOH (1.02 mmol)and water (1 mL) were added and the reaction mixture was stirred for 4h. Then, the mixture was acidified with aqueous HCl (1M, 1.5 ml),concentrated, concentrated and the residue purified by columnchromatography (SiO₂, DCM/MeOH, 1:0 to 9:1) to yield compound 14c (0.41mmol)

Compound 15a:

Compound 14a (0.11 mmol) was dissolved in dioxane (2 ml); pyridine (0.07mmol) and Boc₂O (0.14 mmol) were added followed by NH₄HCO₃ (0.14 mmol).The resulting mixture was stirred for 8 hrs. Afterwards, the mixture wasconcentrated and purified by column chromatography (SiO₂, DCM/MeOH, 1:0to 9:1) to yield compound 15a (0.06 mmol).

Example 10 Synthesis of Linker-Agent Conjugates

General Procedure for the Coupling of the Cyclization Spacer to theActivated Protected Drug (Step 2):

0.2 mmol activated protected drug, 0.6 mmol of mono-protected spacer,and 0.02 mmol HOBt were dissolved in 2 ml DMF, 1 mmol Et₃N was added andthe mixture was heated at 50° C. for 2 hrs. The mixture was concentratedin vacuo and the crude product purified by column chromatography (SiO₂,DCM/MeOH) to give the cyclization spacer-drug.

General Procedure for Coupling of the Linker to the CyclizationSpacer-Drug Intermediate (Steps 3 and 4):

0.1 mmol cyclization spacer-drug was suspended in 6 ml CHCl₃, thereaction mixture was cooled in ice and 2 ml of TFA was added and themixture stirred for 3 hrs. The mixture was then concentrated in vacuo.The residue was dissolved in 4 ml DMF, the solution cooled in ice and0.13 mmol activated linker (16 or 17) and 1 mmol Et₃N were added. Themixture was stirred for 2 hrs, concentrated in vacuo and the residuepurified by column chromatography (SiO₂, DCM/MeOH, 1:0 to 8:2) to an oilwhich was further purified by preparative reversed phase HPLC(acetonitrile/water+0.1% TFA) and freeze-dried to give construct 18 as apale yellow solid.

Compound 18b, R=Me:

¹H-NMR (400 MHz, DMSO-d₆): δ=0.84 (6H, dd, J=6.4 Hz, 2×CH₃ Val),1.32-1.47 (2H, m, CH₂ Cit), 1.52-1.71 (2H, m, CH₂ Cit), 1.91-1.99 (1H,m, β-H Val), 2.81-3.03 (11H, m, 3×CH₃+NCH₂), 4.36-4.43 (1H, m, H-2),4.45-4.51 (1H, m, α-H), 4.63-4.41 (1H, m, H-2), 4.97-5.08 (3H, m, CH₂PABA+H-1), 5.97 (1H, br s, NH), 6.90 (2H, d, J=8.8 Hz, H-3″), 7.01 (2H,s, CH═CH), 7.14-7.59 (7H, m, ArH+2×NH), 7.67-7.80 (3H, m, ArH), 7.91(2H, d, J=8.8 Hz, H-2″), 8.06 (1H, br s, H-6), 8.33 (H, br s, H-4), 8.81(1H, s, H-3′), 9.55 (1H, s, H-4′), 9.99 (1H, s, NH), 10.20 (1H, s, NH),10.28 (1H, s, OH); MS (ESI): m/z=1257.3 (M+H⁺).

Compound 18c, R=Me:

¹H NMR (400 MHz, DMSO-d₆): δ=0.84 (6H, ddi, Val), 1.28-1.73 (4H, m,CH₂CH₂Cit), 1.95 (1H, m, βHVal), 2.85 (3H, s, MeAr), 2.94 (3H, s, MeN),2.87-3.05 (4H, m, 2×CH₂NMe), 4.05 (2H, t, CH₂O), 4.40 (1H, m, H-1), 4.48(1H, s, CH), 4.67 (1H, m, H-2), 5.05 (3H, m, CH₂O, H-2), 5.40 (2H, br s,NH₂), 5.96 (1H, br s, NH), 6.90 (2H, d, H-3″), 7.01 (2H, s, CH═CH),7.2-7.7 (10H, m, ArH+NH), 7.91 (2H, d, H-2″), 8.05 (1H, s, H-6), 8.33(1H, br s, H-4), 8.77 (1H, s, H-3′), 9.52 (1H, s, H-4′), 10.0 (1H, s,NH), 10.19 (1H, s, NH), 10.25 (1H, s, OH); MS (ESI): m/z=1345.7 (M+H⁺).

Compound 18b, R═(CH₂CH₂O)₂H:

¹H NMR (400 MHz, DMSO-d₆), δ (ppm): δ=0.84 (6H, dd, Val), 1.28-1.73 (4H,m, CH₂CH₂Cit), 1.96 (1H, m, βHVal), 2.85 (3H, s, MeAr), 2.95 (3H, s,MeN), 2.87-3.05 (4H, m, 2×CH₂NMe), 4.00 (2H, t, CH₂O), 4.35 (1H, m,H-1), 4.48 (1H, m, CH), 4.67 (1H, m, H-2), 4.93-5.11 (4H, m, CH₂O, H-2,CH), 5.40 (2H, br s, NH₂), 5.97 (1H, br s, NH), 6.91 (2H, d, H-3″), 7.01(2H, s, CH═CH), 7.2-7.7 (10H, m, ArH+NH), 7.91 (2H, d, H-2″), 8.06 (1H,d, H-6), 8.33 (1H, br s, H-4), 8.80 (1H, s, H-3′), 9.54 (1H, s, H-4′),10.01 (1H, s, NH), 10.20 (1H, s, NH), 10.27 (1H, s, OH); MS (ESI):m/z=1331.7 (M+H⁺).

Compound 18c, R═(CH₂)₄C(NH₂)COOH:

¹H-NMR (400 MHz, DMSO-d₆): δ=0.84 (6H, dd, J=6.4 Hz, 2×CH₃ Val),1.29-1.98 (11H, m, 3×CH₂ Lys+2×CH₂ Cit+(3-H Val), 2.82-3.04 (8H, m,2×CH₃+NCH₂), 4.35-4.42 (1H, m, H-1), 4.45-4.51 (1H, m, α-H), 4.63-4.70(1H, m, H-2), 4.95-5.07 (2H, m, CH₂ PABA), 5.09-5.16 (1H, m, H-2), 5.99(1H, br s, NH), 6.90 (2H, d, J=8.4 Hz, H-3″), 7.01 (2H, s, CH═CH),7.16-7.77 (11H, m, ArH+3×NH), 7.90 (2H, d, J=8.4 Hz, H-2″), 8.05-8.09(1H, m, H-6), 8.19 (3H, br s, NH₂ Lys+OH), 8.31-8.37 (1H, m, H-4), 8.75(1H, br s, H-3′), 9.53 (1H, br s, H-4′), 10.00 (1H, br s, NH),10.16-10.26 (2H, m, NH+OH); MS (ESI): m/z=1460.7 (M+H⁺).

Compound 18d, R═(CH₂CH₂O)₂H:

¹H-NMR (400 MHz, DMSO-d₆): δ=0.84 (6H, dd, Val, J=6.8), 1.30-1.46 (2H,m, CH₂ Cit), 1.52-1.70 (2H, m, CH₂ Cit), 1.91-1.99 (1H, m, (3-H Val),2.80-3.05 (10H, m, ArCH₃+NCH₃+2×NCH₂), 3.97-4.06 (5H, m, 2×CH₂O+H-2),4.20 (2H, d, J=5.6 Hz, NCH₂-triazole), 4.36-4.43 (1H, m, H-1), 4.44-4.52(3H, m, CH₂-triazole+α-H), 4.63-4.71 (1H, m, H-2), 4.94-5.09 (3H, m, CH₂PABA+H-2), 5.98 (1H, br s, NH), 6.90 (2H, d, J=8.8 Hz, H-3″), 6.99 (2H,s, CH═CH), 7.16-7.81 (11H, m, ArH+NH), 7.87 (1H, s, triazole-H), 7.91(2H, d, J=8.8 Hz, H-2″), 8.05-8.09 (1H, m, H-6), 8.33 (H, m, H-4), 8.83(1H, s, H-3′), 9.55 (1H, s, H-4′), 10-00 (1H, s, NH), 10.20 (1H, s, NH),10.28 (1H, s, OH); MS (ESI): m/z=1543.7 (M+H⁺).

Compound 18c, R═(CH₂CH₂O)₂H:

¹H NMR (400 MHz, DMSO-d₆), 6=0.84 (6H, dd, Val), 1.28-1.73 (4H, m,CH₂CH₂Cit), 1.96 (1H, m, βHVal), 2.85 (3H, s, MeAr), 2.94 (3H, s, MeN),2.87-3.05 (4H, m, 2×CH₂NMe), 4.05 (2H, t, CH₂O), 4.40 (1H, m, H-1), 4.48(1H, m, CH), 4.67 (1H, m, H-2), 4.93-5.16 (4H, m, CH₂O, CH, H-2), 5.40(2H, br s, NH₂), 5.96 (1H, br s, NH), 6.90 (2H, d, H-3″), 7.01 (2H, s,CH═CH), 7.2-7.8 (11H, m, ArH+3×NH), 7.90 (2H, d, H-2″), 8.05 (1H, d,H-6), 8.32 (1H, br s, H-4), 8.76 (1H, s, H-3′), 9.51 (1H, s, H-4′),10.01 (1H, s, NH), 10.19 (1H, s, NH), 10.24 (1H, s, OH); MS (ESI):m/z=1419.7 (M+H⁺).

Compound 18a, R═(CH₂CH₂O)₂H:

¹H NMR (400 MHz, DMSO-d₆): δ=0.81 (6H, dd, J=6.4 Hz, 2×CH₃), 1.30-1.47(2H, m, CH₂ Cit), 1.53-1.71 (2H, m, CH₂ Cit), 1.89-1.99 (1H, m, (3-HVal), 2.82-3.05 (8H, m, 2×CH₃+NCH₂), 3.96-4.02 (1H, m), 4.15-4.23 (1H,m), 4.33-4.40 (1H, m, H-1), 4.44-4.52 (1H, m, α-H), 4.63-4.71 (1H, m,H-2), 4.94-5.12 (3H, m, CH₂+H-2), 5.96 (1H, br s, NH), 6.90 (2H, d,J=8.4 Hz, H-3″), 7.01 (2H, s, CH═CH), 7.10-7.54 (7H, m, 5 ArH+2×NH),7.66-7.81 (3H, m, ArH), 7.91 (2H, d, J=8.8 Hz, H-2″), 8.00-8.05 (1H, m,H-6), 8.29-8.37 (1H, m, H-4), 8.79 (1H, br s, H-3′), 9.54 (1H, br s,H-4′), 9.91-10.00 (1H, m, NH), 10.20 (1H, br s, OH), 10.27 (1H, s, NH);MS (ESI): m/z=1287.7 (M+H⁺).

Compound 18b, R═(CH₂)₃C(O)NH₂:

¹H-NMR (400 MHz, DMSO-d₆): δ=0.83 (6H, dd, J=6.4 Hz, 2×CH₃ Val),1.25-2.15 (9H, m, 2×CH₂ butyramide+2×CH₂ Cit+(3-H Val), 2.82-3.04 (8H,m, 2×CH₃+NCH₂), 3.18-3.98 (15H, m, 2×H-10+6×CH₂+α-H), 4.34-4.42 (1H, m,H-1), 4.46-4.54 (1H, m, α-H), 4.63-4.71 (1H, m, H-2), 4.93-5.07 (3H, m,CH₂ PABA+H-2), 5.98 (1H, br s, NH), 6.76 (1H, br s, NH), 6.91 (2H, d,J=8.8 Hz, H-3″), 7.00 (2H, s, CH═CH), 7.13-7.58 (8H, m, ArH+2×NH),7.65-7.83 (3H, m, ArH), 7.91 (2H, d, J=8.4 Hz, H-2″), 8.04-8.09 (1H, m,H-6), 8.28-8.37 (1H, m, H-4), 8.89 (1H, br s, H-3′), 9.60 (1H, br s,H-4′), 9.93-10.02 (1H, m, NH), 10.34 (1H, br s, NH); MS (ESI):m/z=1328.3 (M+H⁺).

Compound 18b, R═(CH₂)₄C(NH₂)COOH:

¹H-NMR (400 MHz, DMSO-d₆): δ=0.83 (6H, dd, J=6.4 Hz, 2×CH₃ Val),1.30-1.98 (11H, m, 3×CH₂ Lys+2×CH₂ Cit+(3-H Val), 2.82-3.04 (8H, m,2×CH₃+NCH₂), 4.33-4.42 (1H, m, H-1), 4.45-4.51 (1H, m, α-H), 4.62-4.70(1H, m, H-2), 4.95-5.07 (2H, m, CH₂ PABA), 5.10-5.16 (1H, m, H-2), 5.98(1H, br s, NH), 6.90 (2H, d, J=8.8 Hz, H-3″), 7.01 (2H, s, CH═CH),7.13-7.77 (11H, m, ArH+3×NH), 7.90 (2H, d, J=8.8 Hz, H-2″), 8.05-8.09(1H, m, H-6), 8.19 (3H, br s, NH₂ Lys+OH), 8.31-8.37 (1H, m, H-4), 8.73(1H, br s, H-3′), 9.51 (1H, br s, H-4′), 10.00 (1H, br s, NH),10.17-10.25 (2H, m, NH+OH); MS (ESI): m/z=1372.6 (M+H¹).

Compound 18b, R═(CH₂)₃COOH:

¹H NMR (400 MHz, DMSO-d₆), 6=0.83 (6H, dd, Val), 1.28-1.73 (4H, m,CH₂CH₂Cit), 1.74-2.02 (2H, m, CH₂), 1.95 (1H, m, βHVal), 2.19-2.39 (2H,m, CH₂), 2.85 (3H, s, MeAr), 2.94 (3H, s, MeN), 2.87-3.05 (4H, m,2×CH₂NMe), 4.00 (2H, m, CH₂O), 4.40 (1H, m, H-1), 4.48 (1H, m, CH), 4.67(1H, m, H-2), 4.93-5.18 (4H, m, CH₂O, CH, H-2), 5.40 (2H, br s, NH₂),5.96 (1H, br s, NH), 6.90 (2H, d, H-3″), 7.01 (2H, s, CH═CH), 7.2-7.8(10H, m, ArH+NH), 7.91 (2H, d, H-2″), 8.06 (1H, d, H-6), 8.33 (1H, br s,H-4), 8.76 (1H, s, H-3′), 9.51 (1H, s, H-4′), 10.01 (1H, s, NH), 10.19(1H, s, NH), 10.24 (1H, s, OH); MS (ESI): m/z=1329.8 (M+H¹).

Compound 18c, R═(CH₂)₃COOH:

¹H NMR (400 MHz, DMSO-d₆), 6=0.84 (6H, dd, Val), 1.30-1.73 (4H, m,CH₂CH₂Cit), 1.74-2.02 (2H, m, CH₂), 1.95 (1H, m, βHVal), 2.19-2.39 (2H,m, CH₂), 2.85 (3H, s, MeAr), 2.94 (3H, s, MeN), 2.87-3.05 (4H, m,2×CH₂NMe), 4.05 (2H, m, CH₂O), 4.40 (1H, m, H-1), 4.48 (1H, m, CH), 4.67(1H, m, H-2), 4.95-5.16 (4H, m, CH₂O, CH, H-2), 5.40 (2H, br s, NH₂),5.96 (1H, br s, NH), 6.90 (2H, d, H-3″), 7.01 (2H, s, CH═CH), 7.2-7.8(9H, m, ArH+NH), 7.91 (2H, d, H-2″), 8.05 (1H, d, H-6), 8.34 (1H, br s,H-4), 8.77 (1H, s, H-3′), 9.52 (1H, s, H-4′), 10.01 (1H, s, NH), 10.19(1H, s, NH), 10.24 (1H, s, OH); MS (ESI): m/z=1417.9 (M+H⁺).

Compound 18b, R═(CH₂)₃NHC(O)NH₂:

¹H-NMR (400 MHz, DMSO-d₆): δ=0.79 (6H, dd, Val), 1.25-1.8 (6H, m,3×CH₂), 1.91 (1H, m, βHVal), 2.80 (3H, s, MeAr), 2.90 (3H, s, MeN), 2.95(4H, m, 2×CH₂N), 4.34 (1H, m, H-1), 4.61 (1H, m, H-2), 4.94 (1H, m,H-2), 4.99 (2H, s, CH₂O), 5.32 (4H, br s, 2×NH₂), 5.91 (1H, br s, NH),5.98 (1H, br s, NH), 6.86 (2H, d, H-3″), 6.95 (2H, s, CH═CH), 7.2-7.7(10H, m, ArH+2×NH), 7.86 (2H, d, H-2″), 8.02 (1H, d, H-6), 8.28 (1H, brs, H-4), 8.82 (1H, s, H-3′), 9.54 (1H, s, H-4′), 9.95 (1H, s, NH), 10.15(1H, s, NH), 10.27 (1H, s, OH); MS (ESI): m/z=1343.7 (M+H¹).

Compound 18c, R═(CH₂)₃NHC(O)NH₂:

¹H-NMR (400 MHz, DMSO-d₆): δ=0.79 (6H, dd, Val), 1.25-1.8 (6H, m,3*CH₂), 1.91 (1H, m, βHVal), 2.80 (3H, s, MeAr), 2.90 (3H, s, MeN), 2.95(4H, m, 2×CH₂N), 4.34 (1H, m, CH), 4.45 (1H, m, CH), 4.61 (1H, m, H-2),4.94 (1H, m, H-2), 4.99 (2H, s, CH₂O), 5.32 (4H, br s, 2×NH₂), 5.91 (1H,br s, NH), 5.98 (1H, br s, NH), 6.86 (2H, d, H-3″), 6.95 (2H, s, CH═CH),7.2-7.7 (10H, m, ArH+2×NH), 7.86 (2H, d, H-2″), 8.02 (1H, d, H-6), 8.28(H, br s, H-4), 8.82 (1H, s, H-3′), 9.54 (1H, s, H-4′), 9.95 (1H, s,NH), 10.15 (1H, s, NH), 10.27 (1H, s, OH); MS (ESI): m/z=1431.5 (M+H⁺).

Compound 18d, R═(CH₂)₃NHC(O)NH₂:

¹H-NMR (400 MHz, DMSO-d₆): δ=0.83 (6H, dd, Val), 1.25-1.8 (6H, m,3×CH₂), 1.95 (1H, m, βHVal), 2.85 (3H, s, MeAr), 2.94 (3H, s, MeN), 2.95(4H, m, 2×CH₂N), 4.20 (2H, d, NCH₂), 4.39 (1H, m, CH), 4.48 (3H, m,CH+CH₂), 4.67 (1H, m, H-2), 5.02 (3H, m, H-2, CH₂O), 5.41 (4H, br s,2×NH₂), 5.97 (2H, br s, 2×NH), 6.91 (2H, d, H-3″), 6.99 (2H, s, CH═CH),7.2-7.7 (11H, m, ArH+3×NH), 7.76 (1H, s, triazole-H), 7.88 (2H, d,H-2″), 8.08 (1H, d, H-6), 8.33 (H, br s, H-4), 8.84 (1H, s, H-3′), 9.57(1H, s, H-4′), 10.00 (1H, s, NH), 10.20 (1H, s, NH), 10.30 (1H, s, OH);MS (ESI): m/z=1556.2 (M+H⁺).

Compound 18b, R═(CH₂CH₂O)₃H:

¹H-NMR (400 MHz, DMSO): δ=0.83 (6H, dd, J=6.5 Hz, J=15.8 Hz, Val),1.3-1.7 (4H, m), 1.96 (1H, m), 2.90 (8H, m), 3.00-4.00 (6H, m), 4.44(2H, m), 4.67 (1H, m), 6.69 (1H, s), 5.04 (2H, d, J=9.8 Hz), 5.40 (1H,br s), 5.97 (1H, s, NH), 6.9 (2H, d, J=8.3 Hz), 7.00 (2H, s, CH═CH),7.13-7.61 (7H, m), 7.75 (3H, m) 7.91 (2H, d, J=8.3 Hz), 8.06 (1H, m),8.33 (1H, s), 8.83 (1H, s), 9.56 (1H, s), 9.97 (1H, s), 10.20 (1H, s),10.30 (1H, s); MS (ESI): m/z=1376 (M+H′).

Compound 22: Compound 22 was prepared analogously starting with thecorresponding activated drug. ¹H-NMR (400 MHz, DMSO-d₆): δ=0.84 (6H, dd,J=6.4 Hz, 2×CH₃ Val), 1.30-1.47 (2H, m, CH₂ Cit), 1.52-1.71 (2H, m, CH₂Cit), 1.91-1.99 (1H, m, (3-H Val), 2.82-3.03 (8H, m, 2×CH₃+NCH₂),4.18-4.22 (2H, m, CH₂O), 4.35-4.43 (1H, m, H-1), 4.46-4.52 (1H, m, α-H),4.63-4.71 (1H, m, H-2), 4.94-5.08 (3H, m, CH₂ PABA+H-2), 5.97 (1H, br s,NH), 7.01 (2H, s, CH═CH), 7.09-7.20 (4H, m, H-3″+2 ArH), 7.22-7.58 (5H,m, 3 ArH+2×NH), 7.69-7.82 (3H, m, ArH), 8.00 (2H, d, J=8.8 Hz, H-2″),8.03-8.08 (1H, br s, H-6), 8.28-8.37 (1H, m, H-4), 8.83 (1H, br s,H-3′), 9.57 (1H, br s, H-4′), 9.93-10.02 (1H, m, NH), 10.39 (1H, s, NH);MS (ESI): m/z=1509.6 (M+H⁺).

Compound 23:

Compound 23 was prepared analogously starting with the correspondingactivated drug. ¹H-NMR (400 MHz, DMSO): δ=0.80 (6H, dd, J=6.5 Hz, J=15.9Hz, 2×CH₃), 1.3-1.7 (4H, m), 1.93 (1H, m), 2.9 (11H, m), 3.00-4.50 (10H,m), 5.03 (3H, m), 5.40 (1H, br s), 5.94 (1H, s, NH), 6.98 (2H, s,CH═CH), 7.10-7.85 (11H, m), 8.03 (3H, m), 8.32 (1H, m) 8.76 (1H, s),9.50 (1H, s), 9.78 (1H, s), 9.98 (1H, br s), 10.51 (1H, br s); MS (ESI):m/z=1457 (M+H⁺).

Compound 26:

¹H-NMR (400 MHz, DMSO-d₆): δ=0.83 (6H, dd, J=6.8 Hz, 2×CH₃), 1.30-1.47(2H, m, CH₂ Cit), 1.54-1.71 (2H, m, CH₂ Cit), 1.90-2.00 (1H, m, (3-HVal), 2.82-3.03 (10H, m, ArCH₃+NCH₃+2×NCH₂), 3.19 (3H, s, OCH₃),3.97-4.03 (2H, m, NCH₂-triazole), 4.37-4.52 (2H, m, H-1+α-H), 4.64-4.69(3H, m, H-2+CH₂), 4.94-5.13 (3H, m, CH₂ PABA+H-2), 5.96 (1H, br s, NH),7.01 (2H, s, CH═CH), 7.14-7.58 (7H, m, 5 ArH+3×NH), 7.72-7.84 (3H, m,ArH), 8.04-8.07 (1H, m, H-6), 8.29-8.38 (H, m, H-4), 8.74-8.80 (2H, s,H-3′+triazole-H), 9.53 (1H, s, H-4′), 10-00 (1H, s, NH), 10.85 (1H, s,NH); MS (ESI): m/z=1496.9 (M+H⁺).

Example 11 Synthesis of Compound 19

0.21 g (0.85 mmol) N-Boc-N-methyl-N-methyl-1,2-diamino-1-methylpropane,0.45 g (0.64 mmol) 20 and 8 mg HOBt were dissolved in 5 ml DMF. 0.75 mlDiPEA was added and the reaction mixture stirred at RT overnight. Themixture was concentrated in vacuo and purified by column chromatography(SiO₂, DCM/MeOH, 1:0 to 20:1) to give 0.50 g (100%) of intermediate.This material was suspended in 4 ml CHCl₃ and cooled in ice. 2 ml TFAwas added and the reaction mixture was stirred for 2 hrs at 0° C. andthen concentrated in vacuo. To this material in 6 ml DMF, 0.11 g (0.15mmol) activated MOM-protected drug, 2 mg HOBt and 0.2 ml (1.2 mmol) Et₃Nwere added and the mixture was heated at 50° C. for 3 hrs. The mixturewas concentrated in vacuo and purified by column chromatography (SiO₂,DCM/MeOH, 1:0 to 75:15) to give 0.14 g (72%) material. This wassuspended in 6 ml CHCl₃ cooled in ice. 0.5 ml of TFA was added and themixture was stirred for 2 hrs and then concentrated in vacuo. Thismaterial and 0.045 g (0.17 mmol) maleimide-alkyne linker were dissolvedin 2 ml DMF. 0.6 mL of a solution of 0.025 g (0.1 mmol) CuSO₄, 0.022 g(0.11 mmol) 1,10-phenanthroline and 0.022 g (0.11 mmol) sodium ascorbatein 1 ml acetonitrile/water (1:2) was added to the reaction mixture andthe mixture was stirred for 2.5 hrs. The mixture was acidified withacetic acid and concentrated in vacuo. The crude product was purified bycolumn chromatography (SiO₂, DCM/MeOH, 1:0 to 8:2) to give 0.026 g (16%)material which was further purified by preparative reversed phase HPLCand freeze dried to give 0.015 g of 19. ¹H-NMR (400 MHz, DMSO-d₆):δ=0.79 (6H, dd, Val), 1.34 (3H, br s, gem-Me₂), 1.38 (3H, br s,gem-Me₂), 1.3-1.7 (4H, m, CH₂CH₂Cit), 1.90 (1H, m, βHVal), 2.80 (3H, s,MeAr), 2.91 (3H, s, MeN). 3.11 (3H, br s, MeN), 3.95 (2H, t, CH₂O), 3.98(2H, t, CH₂O), 4.15 (2H, d, NCH₂-triazole), 4.30 (1H, m, H-1), 4.43 (2H,t, CH₂-triazole), 4.61 (1H, m, H-2), 5.00 (2H, s, CH₂O), 5.04 (1H, m,H-2), 5.37 (2H, br s, NH₂), 5.92 (1H, br s, NH), 6.85 (2H, d, H-3″),6.94 (2H, s, CH═CH), 7.2-7.7 (11H, m, ArH+3×NH), 7.81 (1H, s,triazole-H), 7.84 (2H, d, H-2″), 8.03 (1H, d, H-6), 8.27 (H, br s, H-4),8.73 (1H, s, H-3′), 9.48 (1H, s, H-4′), 9.98 (1H, s, NH), 10.15 (1H, s,NH), 10.21 (1H, s, OH); MS (ESI): m/z=1497.6 (M+H⁺).

Compound 21:

The same procedure was followed as for compound 19. ¹H-NMR (400 MHz,DMSO-d₆): δ=0.83 (6H, dd, Val), 1.3-2.1 (3Hm,cyclohexyl+CH₂CH₂Cit+βHVal), 2.84 (3H, s, MeAr), 3.01 (8H, m, 2×MeNCH),3.98 (4H, m, 2×CH₂O), 4.20 (2H, d, NCH₂-triazole), 4.36 (1H, m, H-1),4.48 (2H, t, CH₂-triazole), 4.65 (1H, m, H-2), 5.04 (1H, m, H-2), 5.06(2H, s, CH₂O), 5.39 (2H, br s, NH₂), 5.96 (1H, br s, NH), 6.90 (2H, d,H-3″), 6.99 (2H, s, CH═CH), 7.2-7.7 (11H, m, ArH+3×NH), 7.87 (1H, s,triazole-H), 7.91 (2H, d, H-2″), 8.06 (1H, d, H-6), 8.36 (H, br s, H-4),8.80 (1H, s, H-3′), 9.55 (1H, s, H-4′), 9.97 (1H, s, NH), 10.20 (1H, s,NH), 10.27 (1H, s, OH); MS (ESI): m/z=1523.6 (M+H⁺).

Example 12 Synthesis of Maleimide-Peptide Linkers

Compound 25a:

¹H-NMR (300 MHz, DMSO-d₆): δ=0.83 (6H, dd, J=6.9 Hz, 2×CH₃ Val),1.30-1.51 (2H, m, CH₂ Cit), 1.53-1.76 (2H, m, CH₂ Cit), 1.90-2.01 (1H,m, (3-H Val), 2.89-3.08 (2H, m, CH₂ Cit), 3.62 (2H, t, J=5.4 Hz, CH₂),3.83-3.88 (1H, m, α-H), 3.97-4.05 (1H, m), 4.16-4.24 (1H, m), 4.35-4.43(1H, m, α-H), 5.25 (2H, s, CH₂), 5.41 (2H, s, NH₂), 5.95-5.99 (1H, m,NH), 7.02 (2H, s, HC═CH), 7.11-7.14 (1H, m, NH), 7.40 (2H, d, J=8.7 Hz,Ar—H), 7.54-7.59 (2H, m, Ar—H), 7.64 (2H, d, J=8.4 Hz, Ar—H), 8.03-8.06(1H, m, NH), 8.29-8.33 (2H, m, Ar—H), 10.08 (1H, s, NH); MS (ESI):m/z=712.5 (M+H⁺).

Compound 25b:

¹H-NMR (300 MHz, DMSO-d₆): δ=0.85 (6H, dd, J=6.9 Hz, 2×CH₃ Val),1.32-1.50 (2H, m, CH₂ Cit), 1.54-1.76 (2H, m, CH₂ Cit), 1.91-2.02 (1H,m, (3-H Val), 2.90-3.09 (2H, m, CH₂), 3.49-3.60 (6H, m, CH₂), 3.87-3.92(1H, m, α-H), 3.97-4.06 (2H, m, CH₂), 4.37-4.46 (1H, m, α-H), 5.24 (2H,s, CH₂), 5.41 (2H, s, NH₂), 5.95-6.00 (1H, m, NH), 7.01 (2H, s, HC═CH),7.15-7.18 (1H, m, NH), 7.41 (2H, d, J=8.7 Hz, Ar—H), 7.54-7.59 (2H, m,Ar—H), 7.64 (2H, d, J=8.4 Hz, Ar—H), 8.07-8.10 (1H, m, NH), 8.29-8.33(2H, m, Ar—H), 10.11 (1H, s, NH); MS (ESI): m/z=756.5 (M+H⁺).

Compound 25c:

¹H-NMR (300 MHz, CDCl₃/CD₃OD) δ=0.98 (6H, dd, J=6.9 Hz, J=14.4 Hz, CH₃),1.59 (2H, m), 1.76 (1H, m), 1.94 (1H, m), 2.13 (1H, m), 3.08-3.28 (2H,m) 3.54-3.76 (10H, m, CH₂O), 4.00 (1H, d, J=6.3 Hz), 4.20 (2H, m), 4.57(1H, m), 5.27 (2H, s, OCH₂Ar), 6.78 (2H, s, CH═CH), 7.42 (4H, m, ArH),7.65 (2H, d, J=8.5 Hz), 8.30 (2H, d, J=9.2 Hz).

Compound 25d:

¹H-NMR (400 MHz, CDCl₃/CD₃OD) δ=0.98 (6H, dd, J=6.9 Hz, J=14.4 Hz, CH₃),1.59 (2H, m), 1.76 (1H, m), 1.94 (1H, m), 2.13 (1H, m), 3.08-3.28 (2H,m) 3.57-3.76 (14H, m, CH₂O), 4.00 (1H, m), 4.22 (2H, m), 4.57 (1H, m),5.26 (2H, s, OCH₂Ar), 6.78 (2H, s, CH═CH), 7.42 (4H, m, ArH), 7.66 (2H,d, J=8.3 Hz), 8.29 (2H, d, J=9.3 Hz).

Example 13 Determination of the Cyclization Rate of CyclizationSpacer-Duocarmycin Compounds

Cyclization spacer-duocarmycin compounds in which the cyclization spaceris coupled to the hydroxy group of the DNA-alkylator were prepared fromthe corresponding Boc-protected derivatives such as and similar tocompound 14a by treatment with TFA in DCM for 10 minutes, followed byconcentration. For measurement of the cyclization rate at pH 7.4, thecyclization spacer-duocarmycin compound was dissolved in a mixture of100 mM phosphate buffer pH 7.4 and acetonitrile (60/40) at 25° C. anddisappearance of starting material/appearance of free drug was followedover time by LC/MS (direct injection of reaction mixture). Formeasurement of the cyclization rate at pH 5, the cyclizationspacer-duocarmycin compound was dissolved in a mixture of 100 mM sodiumacetate buffer pH 5 and acetonitrile (60/40) at 37° C. and disappearanceof starting material/appearance of free drug was followed over time byLC/MS (direct injection of reaction mixture). Cyclization rates werecalculated from the LC/MS data. FIG. 2 shows some representative data.All cyclization spacer-duocarmycin compounds depicted in FIG. 2 containthe same duocarmycin compound. CM-Drug1 contains a cyclization spacerused in the prior art. All other cyclization spacer-duocarmycincompounds contain cyclization spacers selected from the ones describedin Examples 1-9. The results presented in FIG. 2 indicate that thecyclization spacers of the present invention can be used to modulate thecyclization rate.

Example 14 Human Plasma Stability of HSA-Conjugated Linker-AgentConjugates

Linker-agent conjugate containing a maleimide group was dissolved inDMSO and added to sodium heparine-stabilized human plasma at 37° C. suchthat the concentration of linker-agent conjugate was 7.5 μM andpercentage DMSO was 2.5%. Linker-agent conjugate reacted in situ withhuman serum albumin within 5 minutes, as indicated by LC/MS. Formationof free drug was followed over time by LC/MS. Plasma samples weretreated with acetonitrile and centrifuged before analysis. FIG. 3presents the human plasma stability data for a representative selectionof linker-agent conjugates of the present invention. All linker-agentconjugates contain the same duocarmycin analog and linkers are coupledto the DNA alkylator moiety, except for LD13, in which the linker isbound to the DNA binder moiety. LD5, LD12, and LD13 contain linkers thathave been used in the prior art and that contain a relatively longlinker as in compound 18d. LD10, LD25, LD26, LD28 and LD29 containsimilar relatively short linkers as in compound 18b and have differentcyclization spacers. LD10 and LD12 only differ in L moiety. The resultsin FIG. 3 demonstrate that conjugates with a relatively short linkerhave a high human plasma stability, which generally exceeds that ofcorresponding conjugates with a relatively long linker. Plasma stabilityis furthermore demonstrated to be affected by the cyclization spacer.

Example 15 Single Dose Efficacy Study in nu/nu Mice Bearing an N87Xenograft

Preparation of ADCs:

Trastuzumab (50 mg, 10 mg/mL) was reduced using 1.1 molar equivalents ofTCEP by incubating at 20° C. for 90 minutes to generate 2 free thiolsper mAb. The incorporation ratio was confirmed via the Ellman's assay.Linker-agent conjugate dissolved in DMSO was added to the reducedantibody solution dropwise at a ratio of 1.3 molar equivalents per freethiol such that the final concentration of DMSO was 10%. After mixingfor a further 50 minutes at 20° C., the reaction was quenched by theaddition of one molar equivalent of N-acetylcysteine per linker-agentconjugate. After quenching, the conjugate was desalted into PBS pH 7.4buffer and then purified using a 5 ml r-Protein A column, with theproduct collected off this column being desalted into presentationbuffer (same buffer as in commercially available Herceptin). The productwas filtered to 0.2 μm and characterized for aggregate (SEC),drug-to-antibody ratio (UV, 280 nm vs 335 nm), and free linker-agentconjugate (LC/MS). ADCs used in the efficacy study described belowcontain a representative selection of linker-agent conjugates of thepresent invention and have an average drug-to-antibody ratio ofapproximately 2.

Efficacy Study:

7-8 week old female nu/nu mice were implanted with N87 tumor fragmentssubcutaneously. Treatment started 19 days after implant when the meanestimated tumor mass for all groups was 130 mg. ADC groups contained 6animals per group; control groups contained 4 animals per group. Animalswere treated with a single dose of 12 mg ADC/kg (0.2 mL/20 g forvehicle; 12 mg mAb/kg for trastuzumab) on day 0. Animals were euthanizedwhen the tumor reached a mass of 1,000 mg. FIG. 4A shows the averagetumor burden for each group. A line stops after a first animal in thecorresponding group has been euthanized due to tumor burden. Figure Bshows the average body weight change for each group. Figure C shows thesurvival percentage in each group. ADC5 is based on a linker-agent thatcontains a relatively long linker as in 18d, whereas the ADCs based onthe other linker-agents contain a relatively short linker as in 18b.ADC30 contains a different drug than the other ADCs, but has the samelinker as ADC28. ADC5 and ADC28 only differ in L moiety. The results inFIG. 4 demonstrate that conjugates that contain a relatively shortlinker have a better efficacy than the corresponding conjugate with arelatively long linker. Both the nature of the linker and the nature ofthe drug were demonstrated to have an effect on efficacy as well.

Example 16 Cleavage of Quenched Linker-Agent Conjugates by Cathepsin B

Linker-agent conjugate was dissolved in acetonitrile/water and 5equivalents of N-acetylcysteine were added. The reaction mixture wasincubated for 1 h at RT and subsequently freeze-dried to give thequenched linker-agent conjugate. Quenched linker-agent conjugatedissolved in DMSO was added to a 5 μg/mL solution of cathepsin B (>200U/mg) in sodium acetate buffer pH 5 at 37° C. The final concentration ofquenched linker-agent conjugate was 130 μM. Disappearance of startingmaterial was monitored over time by LC/MS. FIG. 5 presents the cathepsinB cleavage data for a representative selection of quenched linker-agentconjugates of the present invention. LD5, LD12, and LD13 are based onlinker-agents from the prior art and contain linkers that are relativelylong as in 18d, whereas the linkers in the other quenched linker-agentconjugates are as in 18b. The said other quenched linker-agentconjugates primarily differ in cyclization spacer. The results from FIG.5 demonstrate that all quenched linker-agent conjugates are cleavedefficiently by cathepsin B.

REFERENCES

-   1 Boger, D. L.; Johnson, D. S.; Wrasidlo, W. Bioorg. Med. Chem.    Lett. 1994, 4, 631-636.-   2 McGovren, J. P., Clarke, G. L., Pratt, E. A., DeKoning, T. F. J.    Antibiot. 1984, 37, 63-70.-   3 Carter, P.; Smith, L.; Ryan, M. Endocr.-Relat. Cancer 2004, 11,    659-687.-   4 Bagshawe, K. D. Drug Dev. Res. 1995, 34, 220-230.-   5 Melton, R.; Connors, T.; Knox, R. J. S. T. P. Pharma Sciences,    1999, 13-33.-   6 Huber, B. E.; Richards, C. A.; Krenitsky, T. A. Proc. Natl. Acad.    Sci. USA, 1991, 88, 8039-8043.-   7 Bagshawe, K. D.; Springer, C. J.; Searle, F.; Antoniw, P.;    Sharma, S. K.; Melton, R. G.; Sherwood, R. F. Br. J. Cancer, 1988,    58, 700-703.-   8 Duncan, R. Nat. Rev. Drug Discov. 2003, 2, 347-360.-   9 Toki, B. E.; Cerveny, C. G.; Wahl, A. F.; Senter, P. D. J. Org.    Chem., 2002, 67, 1866-1872.-   10 See for some recently disclosed cyclization spacers for example    WO 2005/079398, WO 2005/105154, and WO 2006/012527.-   11 Greenwald, R. B., Choe, Y. H., McGuire, J., Conover, C. D. Adv.    Drug Delivery Rev. 2003, 55, 217-250.-   12 Kingsbury, W. D.; Boehm; J. C.; Mehta, R. J.; Grappel, S. F.;    Gilvarg, C. J. Med. Chem. 1984, 27, 1447-1451.-   13 Greenwald, R. B.; Zhao, H.; Yang, K.; Reddy, P.; Martinez, A. J.    Med. Chem. 2004, 47, 726-734.-   14 (a) Franke, A. E.; Sievers, E. L.; and Scheinberg, D. A. Cancer    Biother. Radiopharm. 2000, 15, 459-476.-   (b) Murray, J. L. Semin. Oncol. 2000, 27, 2564-2570. (c) Breitling,    F., and Dubel, S., Recombinant Antibodies, John Wiley and Sons, New    York, 1998.-   15 Ringsdorf, H. J. Polym. Sci., Polym. Symp. 1975, 51, 135-153.-   16 Elvira, C.; Gallardo, A.; San Roman, J.; Cifuentes, A. Molecules    2005, 10, 114-125.-   17 Remington's Pharmaceutical Science (15th ed., Mack Publishing,    Easton, Pa., 1980) (incorporated by reference in its entirety for    all purposes)-   18 Pettit, G. R.; Burkett, D. D.; Barkoczy, J.; Breneman, G. L.;    Pettit, W. E. Synthesis 1996, 719-725.

1. A compound of formula (III):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein V² is either absent or a functional moiety; each L² isindependently absent or a linking group linking V² to L; each L isindependently absent or a linking group linking L² to one or more V¹and/or Y; each V¹ is independently absent or a conditionally-cleavableor conditionally-transformable moiety, which can be cleaved ortransformed by a chemical, photochemical, physical, biological, orenzymatic process; each Y is independently absent or a self-eliminatingspacer system which is comprised of 1 or more self-elimination spacersand is linked to V¹, optionally L, and one or more Z; each p and q arenumbers representing a degree of branching and are each independently apositive integer; z is a positive integer equal to or smaller than thetotal number of attachment sites for Z; each Z is independently acompound of formula (I), (II), (I′), or (II′):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein DB is a DNA-binding moiety and is selected from the groupconsisting of

R¹ is a leaving group; R², R^(2′), R³, R^(3′), R⁴, R^(4′), R¹², and R¹⁹are independently selected from H, OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN,C(O)NH₂, C(O)H, C(O)OH, halogen, R^(a), SR^(a), S(O)R^(a), S(O)₂R^(a),S(O)OR^(a), S(O)₂OR^(a), OS(O)R^(a), OS(O)₂R^(a), OS(O)OR^(a),OS(O)₂OR^(a), OR^(a), NHR^(a), N(R^(a))R^(b), ⁺N(R^(a))(R^(b))R^(c),P(O)(OR^(a))(OR^(b)), OP(O)(OR^(a))(OR^(b)), SiR^(a)R^(b)R^(c),C(O)R^(a), C(O)OR^(a), C(O)N(R^(a))R^(b), OC(O)R^(a), OC(O)OR^(a),OC(O)N(R^(a))R^(b), N(R^(a))C(O)R^(b), N(R^(a))C(O)OR^(b), andN(R^(a))C(O)N(R^(b))R^(e), wherein R^(a), R^(b), and R^(c) areindependently selected from H and optionally substituted C₁₋₃ alkyl orC₁₋₃ heteroalkyl, or R³+R^(3′) and/or R⁴+R^(4′) are independentlyselected from ═O, ═S, ═NOR¹⁸, ═C(R¹⁸)R^(18′), and ═NR¹⁸, R¹⁸ and R^(18′)being independently selected from H and optionally substituted C₁₋₃alkyl, two or more of R², R^(2′), R³, R^(3′), R⁴, R^(4′), and R¹²optionally being joined by one or more bonds to form one or moreoptionally substituted carbocycles and/or heterocycles; X² is selectedfrom O, C(R¹⁴)(R^(14′)), and NR^(14′), wherein R¹⁴ and R^(14′) have thesame meaning as defined for R⁷ and are independently selected, orR^(14′) and R^(7′) are absent resulting in a double bond between theatoms designated to bear R^(7′) and R^(14′); R⁵, R^(5′), R⁶, R^(6′), R⁷,and R^(7′) are independently selected from H, OH, SH, NH₂, N₃, NO₂, NO,CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(e), SR^(e), S(O)R^(e),S(O)₂R^(e), S(O)OR^(e), S(O)₂OR^(e), OS(O)R^(e), OS(O)₂R^(e),OS(O)OR^(e), OS(O)₂OR^(e), OR^(e), NHR^(e), N(R^(e))R^(f),⁺N(R^(e))(R^(f))R^(g), P(O)(OR^(e))(OR^(f)), OP(O)(OR^(e))(OR^(f)),SiR^(e)R^(f)R^(g), C(O)R^(e), C(O)OR^(e), C(O)N(R^(e))R^(f), OC(O)R^(e),OC(O)OR^(e), OC(O)N(R^(e))R^(f), N(R^(e))C(O)R^(f), N(R^(e))C(O)OR^(f),N(R^(e))C(O)N(R^(f))R^(g), and a water-soluble group, wherein R^(e),R^(f), and R^(g) are independently selected from H and optionallysubstituted (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₁₅ alkyl, C₁₋₁₅heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₁₅ heterocycloalkyl, C₅₋₁₅ aryl, orC₁₋₁₅ heteroaryl, wherein ee is selected from 1 to 1000, X¹³ is selectedfrom O, S, and NR^(f1), and R^(f1) and R^(e1) are independently selectedfrom H and C₁₋₃ alkyl, one or more of the optional substituents inR^(e), R^(f), and/or R^(g) optionally being a water-soluble group, twoor more of R^(e), R^(f), and R^(g) optionally being joined by one ormore bonds to form one or more optionally substituted carbocycles and/orheterocycles, or R⁵+R^(5′) and/or R⁶+R^(6′) and/or R⁷+R^(7′) areindependently selected from ═O, ═S, ═NOR^(e3), ═C(R^(e3))R^(e4), and═NR^(e3), R^(e3) and R^(e4) being independently selected from H andoptionally substituted C₁₋₃ alkyl, or R^(5′)+R^(6′) and/or R^(6′)+R^(7′)and/or R^(7′)+R^(14′) are absent, resulting in a double bond between theatoms designated to bear R^(5′) and R^(6′), and/or R^(6′) and R^(7′),and/or R^(7′) and R^(14′), respectively, two or more of R⁵, R^(5′), R⁶,R⁷, R^(7′), R¹⁴, and R^(14′) optionally being joined by one or morebonds to form one or more optionally substituted carbocycles and/orheterocycles; X¹ is selected from O, S, and NR¹³, wherein R¹³ isselected from H and optionally substituted C₁₋₈ alkyl or C₁₋₈heteroalkyl and not joined with any other substituent; X³ is selectedfrom O, S, C(R¹⁵)R^(15′), —C(R¹⁵)(R^(15′))—C(R¹⁵″)(R^(15′″))—,—N(R¹⁵)—N(R^(15′))—, —C(R¹⁵)(R^(15′))—N(R¹⁵″)—,—N(R¹⁵″)—C(R¹⁵)(R^(15′))—, —C(R¹⁵)(R^(15′))—O—, —O—C(R¹⁵)(R^(15′))—,—C(R¹⁵)(R^(15′))—S—, —S—C(R¹⁵)(R^(15′))—, —C(R¹⁵)═C(R^(15′))—,═C(R¹⁵)—C(R^(15′))═, —N═C(R^(15′))—, ═N—C(R^(15′))=, —C(R¹⁵)═N—,═C(R¹⁵)—N═, —N═N—, ═N—N═, CR¹⁵, N, and NR¹⁵, or in DB1 and DB2 —X³—represents —X^(3a) and X^(3b)—, wherein X^(3a) is connected to X³⁴, adouble bond is present between X³⁴ and X⁴, and X^(3b) is connected toX¹¹, wherein X^(3a) is independently selected from H and optionallysubstituted (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₈ alkyl, or C₁₋₈heteroalkyl and not joined with any other substituent; X⁴ is selectedfrom O, S, C(R¹⁶)R^(16′), NR¹⁶, N and CR¹⁶; X⁵ is selected from O, S,C(R¹⁷)R^(17′), NOR¹⁷, and NR¹⁷, wherein R¹⁷ and R^(17′) areindependently selected from H and optionally substituted C₁₋₈ alkyl orC₁₋₈ heteroalkyl and not joined with any other substituent; X⁶ isselected from CR¹¹, CR¹¹(R^(11′)), N, NR¹¹, O, and S; X⁷ is selectedfrom CR⁸, CR⁸(R^(8′)), N, NR⁸, O, and S; X⁸ is selected from CR⁹,CR⁹(R^(9′)), N, NR⁹, O, and S; X⁹ is selected from CR¹⁰, CR¹⁰(R^(10′)),N, NR¹⁰, O, and S; X¹⁰ is selected from CR²⁰, CR²⁰(R^(20′)), N, NR²⁰, O,and S; X¹¹ is selected from C, CR²¹, and N, or X¹¹-X^(3b) is selectedfrom CR²¹, CR²¹(R^(21′)), N, NR²¹, O, and S; X¹² is selected from C,CR²², and N; X⁶*, X⁷*, X⁸*, X⁹*, X¹⁰*, and X¹¹* have the same meaning asdefined for X⁶, X⁷, X⁸, X⁹, X¹⁰, and X¹¹, respectively, and areindependently selected; X³⁴ is selected from C, CR²³, and N; the ring Batom of X¹¹* in DB6 and DB7 is connected to a ring atom of ring A suchthat ring A and ring B in DB6 and DB7 are directly connected via asingle bond;

means that the indicated bond may be a single bond or a non-cumulated,optionally delocalized, double bond; R⁸, R^(8′), R⁹, R^(9′), R¹⁰,R^(10′), R¹¹, R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′),R²⁰, R^(20′), R²¹, R^(21′), R²², and R²³ are each independently selectedfrom H, OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH,halogen, R^(h), SR^(h), S(O)R^(h), S(O)₂R^(h), S(O)OR^(h), S(O)₂OR^(h),OS(O)R^(h), OS(O)₂R^(h), OS(O)OR^(h), OS(O)₂OR^(h), OR^(h), NHR^(h),N(R^(h))R^(i), N(R^(h))(R^(i))R^(j), P(O)(OR^(h))(OR^(i)),OP(O)(OR^(h))(OR^(i)), SiR^(h)R^(i)R^(j), C(O)R^(h), C(O)OR^(h),C(O)N(R^(h))R^(i), OC(O)R^(h), OC(O)OR^(h), OC(O)N(R^(h))R^(i),N(R^(h))C(O)R^(i), N(R^(h))C(O)OR^(i), N(R^(h))C(O)N(R^(i))R^(j), and awater-soluble group, wherein R^(h), R^(i), and R^(j) are independentlyselected from H and optionally substituted(CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₁₅ alkyl, C₁₋₁₅ heteroalkyl, C₃₋₁₅cycloalkyl, C₁₋₁₅ heterocycloalkyl, C₅₋₁₅ aryl, or C₁₋₁₅ heteroaryl, oneor more of the optional substituents in R^(h), R^(i), and/or R^(j)optionally being a water-soluble group, two or more of R^(h), R^(i) andR^(j) optionally being joined by one or more bonds to form one or moreoptionally substituted carbocycles and/or heterocycles, or R⁸+R^(8′)and/or R⁹+R^(9′) and/or R¹⁰+R^(10′) and/or R¹¹+^(11′) and/or R¹⁵+R^(15′)and/or R^(15″)+R^(15′″) and/or R¹⁶+R^(16′) and/or R²⁰+R^(20′) and/orR²¹+R^(21′) are independently selected from ═O, ═S, ═NOR^(h1),═C(R^(h1))R^(h2), and ═NR^(h1), R^(h1) and R^(h2) being independentlyselected from H and optionally substituted C₁₋₃ alkyl, two or more ofR⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵, R^(15′),R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′), R²¹, R^(21′), R²², andR²³ optionally being joined by one or more bonds to form one or moreoptionally substituted carbocycles and/or heterocycles; R^(8b) andR^(9b) are independently selected and have the same meaning as R⁸,except that they may not be joined with any other substituent; one of R⁴and R^(4′) and one of R¹⁶ and R^(16′) may optionally be joined by one ormore bonds to form one or more optionally substituted carbocycles and/orheterocycles; one of R², R^(2′), R³, and R^(3′) and one of R⁵ and R^(5′)may optionally be joined by one or more bonds to form one or moreoptionally substituted carbocycles and/or heterocycles; a and b areindependently selected from 0 and 1; the DB moiety does not comprise aDA1, DA2, DA1′, or DA2′ moiety; ring B in DB1 is a heterocycle; if X³ inDB1 represents —X^(3a) and X^(3b)— and ring B is aromatic, then twovicinal substituents on said ring B are joined to form an optionallysubstituted carbocycle or heterocycle fused to said ring B; if X³ in DB2represents —X^(3a) and X^(3b)— and ring B is aromatic, then two vicinalsubstituents on said ring B are joined to form an optionally substitutedheterocycle fused to said ring B, an optionally substituted non-aromaticcarbocycle fused to said ring B, or a substituted aromatic carbocyclewhich is fused to said ring B and to which at least one substituent isattached that contains a hydroxy group, a primary amino group, or asecondary amino group, the primary or secondary amine not being a ringatom in an aromatic ring system nor being part of an amide; if ring A inDB2 is a 6-membered aromatic ring, then substituents on ring B are notjoined to form a ring fused to ring B; two vicinal substituents on ringA in DB8 are joined to form an optionally substituted carbocycle orheterocycle fused to said ring A to form a bicyclic moiety to which nofurther rings are fused; ring A in DB9 together with any rings fused tosaid ring A contains at least two ring heteroatoms; and one or more ofX¹, R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′), R¹⁴, R^(14′), R⁸, R^(8′), R⁹,R^(9′), R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″),R¹⁶, R^(16′), R²⁰, R^(20′), R²¹, R^(21′), R²², and R²³ may optionally inaddition be substituted by or be a substituent of formula (V):

wherein each V^(2′), L^(2′), L′, V^(1′), Y′, Z′, p′, q′, and z′ has thesame meaning as defined for V², L², L, V¹, Y, Z, p, q, and z,respectively, and is independently selected, the one or moresubstituents of formula (V) being independently connected via Y′ to oneor more of X¹, R⁵, R^(5′), R⁶, R^(6′), R⁷, R^(7′), R¹⁴, R^(14′), R⁸,R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹, R^(11′), R¹⁵, R^(15′), R^(15″),R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′), R²¹, R^(21′), R²², R²³, and/or toone or more atoms bearing these R substituents; each Z is independentlyconnected to Y through either X¹, an atom in R⁵, R^(5′), R⁶, R^(6′), R⁷,R^(7′), R¹⁴, R^(14′), R⁸, R^(8′), R⁹, R^(9′), R¹⁰, R^(10′), R¹¹,R^(11′), R¹⁵, R^(15′), R^(15″), R^(15′″), R¹⁶, R^(16′), R²⁰, R^(20′),R²¹, R^(21′), R²², R²³, or an atom bearing any of these R substituents;and at least V² or a V¹ is present.
 2. The compound according to claim 1wherein DB is DB1.
 3. The compound according to any one of claims 1 to 2wherein DB is selected from


4. The compound according to any one of the preceding claims wherein DBis selected from


5. The compound according to any one of the preceding claims wherein Zis

or an isomer of one of these, or a mixture of isomers.
 6. The compoundaccording to any one of the preceding claims wherein Z is

or an isomer of one of these, or a mixture of isomers.
 7. The compoundaccording to any one of the preceding claims wherein X¹ is O and Y isconnected to X¹ via an w-amino aminocarbonyl cyclization spacer beingpart of Y.
 8. The compound according to any one of the preceding claimswherein the w-amino aminocarbonyl cyclization spacer being part of Y isselected from

wherein R¹¹⁵ and R¹¹⁶ are independently selected from H, methyl, ethyl,isopropyl, tert-butyl, phenyl, (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1),

wherein dd is selected from 0 to 10, dd′ is selected from 0 and 1, eachR¹²³ is independently selected from H and methyl, ee is selected from 1to 1000, X¹³ is selected from O, S, and NR^(f1), and R^(f1) and R^(e1)are independently selected from H and C₁₋₃ alkyl, and wherein R¹¹⁷,R¹¹⁸, R¹¹⁹, R¹²⁰, R¹²¹, and R¹²² are independently selected from H, OH,SH, NH₂, N₃, NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(z),SR^(z), S(O)R^(z), S(O)₂R^(z), S(O)OR^(z), S(O)₂OR^(z), OS(O)R^(z),OS(O)₂R^(z), OS(O)OR^(z), OS(O)₂OR^(z), OR^(z), NHR^(z), N(R^(z))R^(z1),⁺N(R^(z))(R^(z1))R^(z2), P(O)(OR^(z))(OR^(z1)), OP(O)(OR^(z))(OR^(z1)),C(O)R^(z), C(O)OR^(z), C(O)N(R^(z1))R^(z), OC(O)R^(z), OC(O)OR^(z),OC(O)N(R^(z))R^(z1), N(R^(z1))C(O)R^(z), N(R^(z1))C(O)OR^(z), andN(R^(z1))C(O)N(R^(z2))R^(z), wherein R^(z), R^(z1), and R^(z2) areindependently selected from H and optionally substituted(CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₂₀ alkyl, C₁₋₂₀ heteroalkyl, C₃₋₂₀cycloalkyl, C₁₋₂₀ heterocycloalkyl, C₅₋₂₀ aryl, or C₁₋₂₀ heteroaryl,wherein ee is selected from 1 to 1000, X¹³ is selected from O, S, andNR^(f1), and R^(f1) and R^(e1) are independently selected from H andC₁₋₃ alkyl.
 9. The compound according to any one of the preceding claimswherein the w-amino aminocarbonyl cyclization spacer being part of Y is


10. The compound according to any one of the preceding claims whereinthe spacer system Y is

wherein A is


11. The compound according to any one of the preceding claims wherein V¹contains a substrate that can be cleaved by a proteolytic enzyme,plasmin, a cathepsin, cathepsin B, β-glucuronidase, a galactosidase,prostate-specific antigen (PSA), urokinase-type plasminogen activator(u-PA), a member of the family of matrix metalloproteinases, or anenzyme localized by means of directed enzyme prodrug therapy, such asADEPT, VDEPT, MDEPT, GDEPT, or PDEPT, or wherein V¹ contains a moietythat can be cleaved or transformed through reduction under hypoxicconditions, through reduction by a nitroreductase, or through oxidation.12. The compound according to any one of the preceding claims whereinthe chain of atoms of L linking L² to V¹ consists of less than 19 atoms.13. The compound according to any one of the preceding claims wherein Lis selected from


14. The compound according to any one of the preceding claims wherein L²is


15. The compound according to any one of the preceding claims whereinthe V² moiety is an antibody or an antibody fragment or a derivativethereof.
 16. The compound according to claim 1, which is

or an isomer, or a mixture of isomers, wherein R⁵ is selected from H,methyl and methoxy, R⁶, R⁷, and R¹⁴ are H, DB is DB1, V¹ is selectedfrom valylcitrulline, valyllysine, phenylalanyllysine,alanylphenylalanyllysine, and D-alanylphenylalanyllysine, f is 1 or 2,CL is selected from

L is selected from

q ranges from 1 to 4, and Ab is an antibody or a fragment or derivativethereof.
 17. The compound according to any one of the preceding claimswherein the V² moiety is selected from an anti-CD19 antibody, ananti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, ananti-CD56 antibody, an anti-CD70 antibody, an anti-CD74 antibody, ananti-CD138 antibody, an anti-CLL-1 antibody, an anti-5T4 antibody, ananti-CD303 antibody, an anti-Tag 72 antibody, an anti-Lewis A likecarbohydrate antibody, an anti-EphB3 antibody, an anti-HMW-MAA antibody,an anti-CD38 antibody, an anti-Cripto antibody, an anti-EphA2 antibody,an anti-GPNMB antibody, an anti-integrin antibody, an anti-MN antibody,an anti-Her2 antibody, and an anti-PSMA antibody, or from anepitope-binding functional fragment or derivative of any of these.
 18. Acompound of formula (IV):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein RM is a reactive moiety and L, V¹, Y, Z, p, and z are as definedin claim 1, except that L is now linking RM to one or more V¹ and/or Y,and V¹, Y, and Z may contain protecting groups, and the one or moreV^(2′)-L^(2′) moieties optionally present in Z as defined hereinabovemay optionally and independently be RM′ instead, which is a reactivemoiety, and wherein, if there is more than 1 reactive moiety in (IV),some or all reactive moieties are the same or different.
 19. Thecompound according to claim 18 wherein the reactive moiety RM is

wherein X³⁵ is selected from halide, hydroxy, OC(O)R^(dd), andOC(O)OR^(dd), or C(O)—X³⁵ is an active ester, X³⁶ is selected fromhalide, mesyloxy, triflyloxy, and tosyloxy, and R^(dd) is selected fromoptionally substituted C₁₋₁₀ alkyl, C₁₋₁₀ heteroalkyl, C₃₋₁₀ cycloalkyl,C₁₋₁₀ heterocycloalkyl, C₅₋₁₀ aryl, and C₁₋₁₀ heteroaryl.
 20. Thecompound according to claim 18, which is

or an isomer, or a mixture of isomers, wherein R^(5a) is selected fromH, methyl and methoxy, AZ is

V¹ is selected from valylcitrulline, valyllysine, phenylalanyllysine,alanylphenylalanyllysine, and D-alanylphenylalanyllysine, CL is

and L is selected from


21. A compound of formula (I) or (II):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein DB is a DNA-binding moiety of formula:

R¹ is a leaving group; R², R^(2′), R³, R^(3′), R⁴, R^(4′), R′², and R¹⁹are independently selected from H, OH, SH, NH₂, N₃, NO₂, NO, CF₃, CN,C(O)NH₂, C(O)H, C(O)OH, halogen, R^(a), SR^(a), S(O)R^(a), S(O)₂R^(a),S(O)OR^(a), S(O)₂OR^(a), OS(O)R^(a), OS(O)₂R^(a), OS(O)OR^(a),OS(O)₂OR^(a), OR^(a), NHR^(a), N(R^(a))R^(b), ⁺N(R^(a))(R^(b))R^(c),P(O)(OR^(a)(OR^(b)), OP(O)(OR^(a))(OR^(b)), SiR^(a)R^(b)R^(c),C(O)R^(a), C(O)OR^(a), C(O)N(R^(a))R^(b), OC(O)R^(a), OC(O)OR^(a),OC(O)N(R^(a))R^(b), N(R^(a))C(O)R^(b), N(R^(a))C(O)OR^(b), andN(R^(a))C(O)N(R^(b))R^(c), wherein R^(a), R^(b), and R^(c) areindependently selected from H and optionally substituted C₁₋₃ alkyl orC₁₋₃ hetero alkyl, or R³+R^(3′) and/or R⁴+R^(4′) are independentlyselected from ═O, ═S, ═NOR¹⁸, ═C(R¹⁸)R^(18′), and ═NR¹⁸, R¹⁸ and R^(18′)being independently selected from H and optionally substituted C₁₋₃alkyl, two or more of R², R^(2′), R³, R^(3′), R⁴, R^(4′), and R¹²optionally being joined by one or more bonds to form one or moreoptionally substituted carbocycles and/or heterocycles; X² is selectedfrom O, C(R¹⁴)(R^(14′)), and NR^(14′), wherein R¹⁴ and R^(14′) have thesame meaning as defined for R⁷ and are independently selected, orR^(14′) and R^(7′) are absent resulting in a double bond between theatoms designated to bear R^(7′) and R^(14′); R⁵, R^(5′), R⁶, R^(6′), R⁷,and R^(7′) are independently selected from H, OH, SH, NH₂, N₃, NO₂, NO,CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(e), SR^(e), S(O)R^(e),S(O)₂R^(e), S(O)OR^(e), S(O)₂OR^(e), OS(O)R^(e), OS(O)₂R^(e),OS(O)OR^(e), OS(O)₂OR^(e), OR^(e), NHR^(e), N(R^(e))R^(f),N(R^(e))(R)R^(g), P(O)(OR^(e))(OR^(f)), OP(O)(OR^(e))(OR^(f)),SiR^(e)R^(f)R^(g), C(O)R^(e), C(O)OR^(e), C(O)N(R^(e))R^(f), OC(O)R^(e),OC(O)OR^(e), OC(O)N(R^(e))R^(f), N(R^(e))C(O)R^(f), N(R^(e))C(O)OR^(f),N(R^(e))C(O)N(R^(f))R^(g), and a water-soluble group, wherein R^(e),R^(f), and R^(g) are independently selected from H and optionallysubstituted (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1) C₁₋₁₅ alkyl, C₁₋₁₅heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₁₅ heterocycloalkyl, C₅₋₁₅ aryl, orC₁₋₁₅ heteroaryl, wherein ee is selected from 1 to 1000, X¹³ is selectedfrom O, S, and NR^(f1), and R^(f1) and R^(e1) are independently selectedfrom H and C₁₋₃ alkyl, one or more of the optional substituents inR^(e), R^(f), and/or R^(g) optionally being a water-soluble group, twoor more of R^(e), R^(f), and R^(g) optionally being joined by one ormore bonds to form one or more optionally substituted carbocycles and/orheterocycles, or R⁵+R^(5′) and/or R⁶+R^(6′) and/or R⁷+R^(7′) areindependently selected from ═O, ═S, ═NOR^(e3), ═C(R^(e3))R^(e4), and═NR^(e3), R^(e3) and R^(e4) being independently selected from H andoptionally substituted C₁₋₃ alkyl, or R^(5′)+R^(6′) and/or R^(6′)+R^(7′)and/or R^(7′)+R^(14′) are absent, resulting in a double bond between theatoms designated to bear R^(5′) and R^(6′), and/or R^(6′) and R^(7′),and/or R^(7′) and R^(14′), respectively, two or more of R⁵, R^(5′), R⁶,R^(6′), R⁷, R^(7′), R¹⁴, and R^(14′) optionally being joined by one ormore bonds to form one or more optionally substituted carbocycles and/orheterocycles; X¹ is selected from O, S, and NR¹³, wherein R¹³ isselected from H and optionally substituted C₁₋₈ alkyl or C₁₋₈heteroalkyl and not joined with any other substituent; R⁸, R⁹, R¹⁰, R¹¹,R¹⁵, and R¹⁶ are each independently selected from H, OH, SH, NH₂, N₃,NO₂, NO, CF₃, CN, C(O)NH₂, C(O)H, C(O)OH, halogen, R^(h), SR^(h),S(O)R^(h), S(O)₂R^(h), S(O)OR^(h), S(O)₂OR^(h), OS(O)R^(h), OS(O)₂R^(h),OS(O)OR^(h), OS(O)₂OR^(h), OR^(h), NHR^(h), N(R^(h))R^(i),⁺N(R^(h))(R^(i))R^(j), P(O)(OR^(h))(OR^(i)), OP(O)(OR^(h))(OR^(i)),SiR^(h)R^(i)R^(j), C(O)R^(h), C(O)OR^(h), C(O)N(R^(h))R^(i), OC(O)R^(h),OC(O)OR^(h), OC(O)N(R^(h))R^(i), N(R^(h))C(O)R^(i), N(R^(h))C(O)OR^(i),N(R^(h))C(O)N(R^(i))R^(j), and a water-soluble group, wherein R^(h),R^(i), and R^(j) are independently selected from H and optionallysubstituted (CH₂CH₂O)_(ee)CH₂CH₂X¹³R^(e1), C₁₋₁₅ alkyl, C₁₋₁₅heteroalkyl, C₃₋₁₅ cycloalkyl, C₁₋₁₅ heterocycloalkyl, C₅₋₁₅ aryl, orC₁₋₁₅ heteroaryl, one or more of the optional substituents in R^(h),R^(i), and/or R^(j) optionally being a water-soluble group, two or moreof R^(h), R^(i), and R^(j) optionally being joined by one or more bondsto form one or more optionally substituted carbocycles and/orheterocycles, two or more of R⁸, R⁹, R¹⁰, R¹¹, R¹⁵m and R¹⁶ optionallybeing joined by one or more bonds to form one or more optionallysubstituted carbocycles and/or heterocycles; one of R⁴ and R^(4′) andR¹⁶ may optionally be joined by one or more bonds to form one or moreoptionally substituted carbocycles and/or heterocycles; one of R²,R^(2′), R³, and R^(3′) and one of R⁵ and R^(5′) may optionally be joinedby one or more bonds to form one or more optionally substitutedcarbocycles and/or heterocycles; and a and b are independently selectedfrom 0 and
 1. 22. Use of a compound according to any one of claims 18 to20 for the preparation of a compound of claim
 1. 23. Use of a compoundaccording to any one of claims 1 to 21 for the manufacture of apharmaceutical composition for the treatment or prevention of a tumor ina mammal.
 24. A pharmaceutical composition comprising a compoundaccording to any one of claims 1 to 21 and a pharmaceutically acceptablecarrier.
 25. A process for preparing a pharmaceutical compositioncomprising the step of mixing a compound according to any one of claims1 to 21 with a pharmaceutically acceptable carrier.
 26. A method oftreating or preventing a tumor in a mammal, whereby the method comprisesthe administration of a pharmaceutical composition according to claims23 to 24 or obtained according to the process of claim 25, to the mammalin a therapeutically effective dose.